PSMA-binding agents and uses thereof

ABSTRACT

Prostate-specific membrane antigen (PSMA) binding compounds having radioisotope substituents are described, as well as chemical precursors thereof. Compounds include pyridine containing compounds, compounds having phenylhydrazine structures, and acylated lysine compounds. The compounds allow ready incorporation of radionuclides for single photon emission computed tomography (SPECT) and positron emission tomography (PET) for imaging, for example, prostate cancer cells and angiogenesis.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/211,683, filed Mar. 14, 2014, which is a divisional of U.S.application Ser. No. 13/057,044, filed Feb. 1, 2011, which is a 35U.S.C. §371 U.S. national phase entry of International Application No.PCT/US2009/052456 having an international filing date of Jul. 31, 2009,which claims the benefit of U.S. Provisional Application No. 61/085,462,filed Aug. 1, 2008, and U.S. Provisional Application No. 61/111,791,filed Nov. 6, 2008, the content of each of the aforementionedapplications is herein incorporated by reference in its entirety.

BACKGROUND

Field of the Invention

The present invention relates generally to radioisotope labeled prostatespecific membrane antigen (PSMA) binding compounds, chemical precursorsof radioisotope labeled PSMA binding compounds and imaging methods usingthe radioisotope labeled compounds.

Background

Prostate cancer (PCa) is the second leading cause of cancer-relateddeath in men (1). Only one half of tumors due to PCs are clinicallylocalized at diagnosis and one half of those represent extracapsularspread. Localization of that spread as well as determination of thetotal body burden of PCa have important implications for therapy,particularly as new combination and local therapies become available.Also critically needed are targeted agents that can provide a readout onthe biology of the tumor, with the ability to predict which tumors willlie dormant and which will develop into aggressive, metastatic disease.The current clinical standard for localizing cancer—including PCa—isshifting from the anatomic techniques such as computed tomography (CT)and magnetic resonance (MR) imaging to more physiologically relevantmethods that employ molecular imaging, such as MR spectroscopy, singlephoton emission computed tomography (SPECT) and positron emissiontomography (PET) (2). Such newer methods that utilize molecular imagingmay provide the biological readout necessary for understanding tumorphysiology, enabling more accurate prognosis and therapeutic monitoring.Molecular imaging may provide a way to not only detect tumors in vivo,but also to provide information regarding the biology of the lesion, ifa mechanism-specific agent is used. For example, [¹⁸F]FDHT can be usedto study the androgen receptor status of tumors (3).

Unlike many other cancers, PCa is particularly difficult to detect usingexisting molecular imaging tracers. There are several reasons for this,including the relatively slow growth and metabolic rate of PCa comparedto other malignancies as well as the small size of the organ andproximity to the urinary bladder, into which most radiopharmaceuticalsare eventually excreted.

Because of the relatively low metabolism of PCa, PET with[¹⁸F]fluorodeoxyglucose (FDG-PET) has proved ineffectual for diagnosticimaging of this disease. Other promising, experimentalradiopharmaceuticals for imaging PCa are emerging, including those ofthe choline series (4) (5) (6), radiolabeled acetates (7),anti-1-amino-3-[¹⁸F]fluorocyclobutyl-1-carboxylic acid (anti[¹⁸F]-FACBC)(8) (9), 1-(2-deoxy-2-[¹⁸F]fluoro-L-arabinofuranosyl)-5-methyluracil([¹⁸F]FMAU) (10) and [¹⁸F]fluorodihydrotestosterone ([¹⁸F]FDHT) (3).Each has its benefits and detriments, with no single agent ideal, i.e.,easy to synthesize, little metabolism and demonstrating tumor-specificuptake, in all PCa phenotypes.

Overexpressed on most solid tumor neovasculature (11) as well as inprostate cancer, the prostate-specific membrane antigent (PSMA) isbecoming an attractive target for cancer imaging and therapy (12) (13).PSMA-based agents can report on the presence of this marker, which isincreasingly recognized as an important prognostic determinate in PCa(14). It is also the target for a variety of new PCa therapies (15).ProstaScint™ is an ¹¹¹In-labeled monoclonal antibody against PSMA thatis clinically available for imaging PCa. ProstaScint™ and radiolabeledvariations of this antibody are fraught with long circulation times andpoor target to nontarget tissue contrast, limiting the utility of theseagents (16) (17) (18).

SUMMARY OF THE INVENTION

The present invention satisfies the long standing and unmet need for newtissue-specific compounds for imaging prostate cancer and angiogenesis.The present invention, in particular, provides imaging agents whichdiffer from the prior art in modifications which were not previouslyknown or suggested. Furthermore, the invention provides imaging agentsthat offer better contrast between target tissues and non-targettissues.

The invention relates to compounds having the structure (I) shown below.

wherein Z is tetrazole or CO₂Q; each Q is independently selected fromhydrogen or a protecting group.

In some embodiments of Formula I, m is 0, 1, 2, 3, 4, 5, or 6; R is apyridine ring with the structure

wherein X is fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, chlorine, bromine, a radioisotope of bromine, aradioisotope of astatine, NO₂, NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²),B(OH)₂, —NHNH₂, —NHN═CHR³, —NHNH—CH₂R³; n is 1, 2, 3, 4, or 5; Y is O,S, N(R′), C(O), NR′C(O), C(O)N(R′), OC(O), C(O)O, NR′C(O)NR′,NR′C(S)NR′, NR′S(O)₂, S(CH₂)_(p), NR′(CH₂)_(p), O(CH₂)_(p),OC(O)CHR⁸NHC(O), NHC(O)CHR⁸NHC(O), or a covalent bond; wherein p is 1,2, or 3, R′ is H or C₁-C₆ alkyl, and R⁸ is alkyl, aryl or heteroaryl,each of which may be substituted; R² is C₁-C₆ alkyl; and R³ is alkyl,alkenyl, alkynyl, aryl, or heteroaryl each of which is substituted byfluorine, iodine, a radioisotope of fluorine, a radioisotope of iodine,bromine, a radioisotope of bromine, a radioisotope of astatine, NO₂,NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²), or B(OH)₂.

In some embodiments of Formula I, m is 0, 1, 2, 3, 4, 5, or 6; Y is O,S, N(R′), C(O), NR′C(O), C(O)N(R′), OC(O), C(O)O, NR′C(O)NR′,NR′C(S)NR′, NR′S(O)₂, S(CH₂)_(p), NR′(CH₂)_(p), O(CH₂)_(p),OC(O)CHR⁸NHC(O), NHC(O)CHR⁸NHC(O), or a covalent bond; wherein p is 1,2, or 3, R′ is H or C₁-C₆ alkyl, and R⁸ is alkyl, aryl or heteroaryl,each of which may be substituted; R is

wherein X′ is selected from the group consisting of NHNH₂, —NHN═CHR³,and —NHNH—CH₂R³; wherein R³ is alkyl, alkenyl, alkynyl, aryl, orheteroaryl each of which is substituted by fluorine, iodine, aradioisotope of fluorine, a radioisotope of iodine, chlorine, bromine, aradioisotope of bromide, or a radioisotope of astatine, NO₂, NH₂,N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²), or B(OH)₂; and n is 1, 2, 3, 4, or 5.

In other embodiments of Formula I, m is 4, Y is NR′, and R is

wherein G is O, NR′ or a covalent bond; R′ is H or C₁-C₆ alkyl; p is 1,2, 3, or 4, and R⁷ is selected from the group consisting of NH₂, N═CHR³,NH—CH₂R³, wherein R³ is alkyl alkenyl, alkynyl, aryl, or heteroaryl eachof which is substituted by fluorine, iodine, a radioisotope of fluorine,a radioisotope of iodine, chlorine, bromine, a radioisotope of bromine,a radioisotope of astatine, NO₂, NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²),or B(OH)₂.

Some compounds of the present invention interact with theprostate-specific membrane antigen (PMSA). As a result, when thecompounds comprise a radioisotope, they may be suitable as imagingagents, diagnostic agents, and/or therapeutic agents.

In many cases, the radioisotope used in the compound is short-lived.Therefore, radioisotopically labeled compounds are prepared immediatelyor shortly before use, or only in sufficient quantity foradministration. For this reason, the invention also includes precursorsto radioisotopically labeled compounds, which may be chemicallyconverted into the radioisotopically labeled compounds of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an overlay of the best poses for 3, 6 and 8 with thecrystal ligand, i.e., 3 as it is co-crystallized with PSMA, in thepresence of water molecule in the active site of PSMA (PDB ID: 3D7H).Dark spheres (zinc ions), light sphere (chloride ion).

FIG. 2 shows an overlay of the best poses (3, 6 and 8) with the crystalligand (3) in the absence of water molecule in the active site of PSMA.Dark sphere (zinc ions), light sphere (chloride ion).

FIG. 3 shows [¹²⁵I]3 SPECT-CT in PCa tumor models (4 h postinjection).Note uptake within the PSMA+PIP tumor only. Uptake within kidneys is duein large measure to the specific binding of [¹²⁵I]3 to renal cortex.

FIG. 4 shows [¹⁸F]6 PET coregistered to CT in PCa tumor models (˜100 minpostinjection). Note uptake within the PSMA+PIP tumor only. Uptakewithin kidneys is due in large measure to the specific binding of[¹²⁵I]3 to renal cortex. There is more intense tumor uptake and lessliver seen with this agent than with [¹²⁵I]3.

FIG. 5 shows [¹²⁵I]8 SPECT-CT in PSMA+LNCaP tumors (4 h postinfection).Note intense uptake within tumor. A similar result was obtained forPSMA+PIP but not PSMA-flu tumors (data not shown). There is less renaland liver uptake with this agent than the halobenzoylated analogues,[¹²⁵I]3 and [¹⁸F]6, respectively.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Embodiments of the invention include compounds according to formula I,shown below:

wherein Z is tetrazole or CO₂Q, and each Q is independently selectedfrom hydrogen or a protecting group.

In exemplary embodiments (A), m is 0, 1, 2, 3, 4, 5, or 6, R is apyridine ring selected from the group consisting of

wherein X is fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, chlorine, bromine, a radioisotope of bromine, aradioisotope of astatine, NO₂, NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²),B(OH)₂, —NHNH₂, —NHN═CHR³, —NHNH—CH₂R³; n is 1, 2, 3, 4, or 5; Y is O,S, N(R′), C(O), NR′C(O), C(O)N(R′), OC(O), C(O)O, NR′C(O)NR′,NR′C(S)NR′, NR′S(O)₂, S(CH₂)_(p), NR′(CH₂)_(p), O(CH₂)_(p),OC(O)CHR⁸NHC(O), NHC(O)CHR⁸NHC(O), or a covalent bond; p is 1, 2, or 3,R′ is H or C₁-C₆ alkyl and R⁸ is alkyl, aryl or heteroaryl, each ofwhich may be substituted; R² is C₁-C₆ alkyl; and R³ is alkyl, alkenyl,alkynyl, aryl, or heteroaryl each of which is substituted by fluorine,iodine, a radioisotope of fluorine, a radioisotope of iodine, chlorine,bromine, a radioisotope of bromine, or a radioisotope of astatine, NO₂,NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²), or B(OH)₂; or a pharmaceuticallyacceptable salt thereof.

In other embodiments (B), m is 0, 1, 2, 3, 4, 5, or 6; Y is O, S, N(R′),C(O), NR′C(O), C(O)N(R′), OC(O), C(O)O, NR′C(O)NR′, NR′C(S)NR′,NR′S(O)₂, S(CH₂)_(p), NR′(CH₂)_(p), O(CH₂)_(p), OC(O)CHR⁸NHC(O),NHC(O)CHR⁸NHC(O), or a covalent bond; p is 1, 2, or 3; R′ is H or C₁-C₆alkyl; R⁸ is alkyl, aryl or heteroaryl, each of which may besubstituted; R is

wherein X′ is selected from the group consisting of NHNH₂, —NHN═CHR³,and —NHNH—CH₂R³; wherein R³ is alkyl, alkenyl, alkynyl, aryl, orheteroaryl each of which is substituted by fluorine, iodine, aradioisotope of fluorine, a radioisotope of iodine, chlorine, bromine, aradioisotope of bromine, or a radioisotope of astatine; NO₂, NH₂,N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²), or B(OH)₂; R² is C₁-C₆ alkyl; n is 1,2, 3, 4, or 5; or a pharmaceutically acceptable salt thereof.

In yet other embodiments (C), m is 4; Y is NR′; and R is

wherein G is O, NR′ or a covalent bond; R′ is H or C₁-C₆ alkyl; p is 1,2, 3, or 4, and R⁷ is selected from the group consisting of NH₂, N═CHR³,NH—CH₂R³, wherein R³ is alkyl, alkenyl, alkynyl, aryl, or heteroaryleach of which is substituted by fluorine, iodine, a radioisotope offluorine, a radioisotope of iodine, bromine, a radioisotope of bromine,or a radioisotope of astatine; NO₂, NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃,Hg(R²), or B(OH)₂; R² is C₁-C₆ alkyl; or a pharmaceutically acceptablesalt thereof.

The compounds herein described may have one or more asymmetric centersor planes. Compounds of the present invention containing anasymmetrically substituted atom may be isolated in optically active orracemic terms. It is well known in the art how to prepare opticallyactive forms, such as by resolution of racemic forms (racemates), byasymmetric synthesis, or by synthesis from optically active startingmaterials. Resolution of the racemates can be accomplished, for example,by conventional methods such as crystallization in the presence of aresolving agent, or chromatography, using, for example a chiral HPLCcolumn. Many geometric isomers of olefins, C═N double bonds, and thelike can also be present in the compounds described herein, and all suchstable isomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral (enantiomeric and diastereomeric), andracemic forms, as well as all geometric isomeric forms of a structureare intended, unless the specific stereochemistry or isomeric form isspecifically indicated.

The compounds herein described may have one or more charged atoms. Forexample, the compounds may be zwitterionic, but may be neutral overall.Other embodiments may have one or more charged groups, depending on thepH and other factors. In these embodiments, the compound may beassociated with a suitable counter-ion. It is well known in the art howto prepare salts or exchange counter-ions. Generally, such salts can beprepared by reacting free acid forms of these compounds with astoichiometric amount of the appropriate base (such as Na, Ca, Mg, or Khydroxide, carbonate, bicarbonate, or the like), or by reacting freebase forms of these compounds with a stoichiometric amount of theappropriate acid. Such reactions are typically carried out in water orin an organic solvent, or in a mixture of the two. Counter-ions may bechanged, for example, by ion-exchange techniques such as ion-exchangechromatography. All zwitterions, salts and counter-ions are intended,unless the counter-ion or salt is specifically indicated. In certainembodiments, the salt or counter-ion may be pharmaceutically acceptable,for administration to a subject. Pharmaceutically acceptable salts arediscussed later.

When any variable occurs more than one time in any constituent orformula for a compound, its definition at each occurrence is independentof its definition at every other occurrence. Thus, for example, if agroup is shown, to be substituted with (X)_(n), where n is 1, 2, 3, 4,or 5, then said group may optionally be substituted with up to five Xgroups and each occurrence is selected independently from the definitionof X. Also, combinations of substituents and/or variables arepermissible only if such combinations result in stable compounds.

As indicated above, various substituents of the various formulae are“substituted” or “may be substituted.” The term “substituted,” as usedherein, means that any one or more hydrogens on the designated atom orgroup is replaced with a substituent, provided that the designatedatom's normal valence is not exceeded, and that the substitution resultsin a stable compound. When a substituent is oxo (keto, i.e., =0), then 2hydrogens on an atom are replaced. The present invention is intended toinclude all isotopes (including radioisotopes) of atoms occurring in thepresent compounds. When the compounds are substituted, they may be sosubstituted at one or more available positions, typically 1, 2, 3 or 4positions, by one or more suitable groups such as those disclosedherein. Suitable groups that may be present on a “substituted” groupinclude e.g., halogen; cyano; hydroxyl; nitro; azido; amino; alkanoyl(such as a C₁-C₆ alkanoyl group such as acyl or the like); carboxamido;alkyl groups (including cycloalkyl groups, having 1 to about 8 carbonatoms, for example 1, 2, 3, 4, 5, or 6 carbon atoms); alkenyl andalkynyl groups (including groups having one or more unsaturated linkagesand from 2 to about 8, such as 2, 3, 4, 5 or 6, carbon atoms); alkoxygroups having one or more oxygen linkages and from 1 to about 8, forexample 1, 2, 3, 4, 5 or 6 carbon atoms; aryloxy such as phenoxy;alkylthio groups including those having one or more thioether linkagesand from 1 to about 8 carbon atoms, for example 1, 2, 3, 4, 5 or 6carbon atoms; alkylsulfinyl groups including those having one or moresulfinyl linkages and from 1 to about 8 carbon atoms, such as 1, 2, 3,4, 5, or 6 carbon atoms; alkylsulfonyl groups including those having oneor more sulfonyl linkages and from 1 to about 8 carbon atoms, such as 1,2, 3, 4, 5, or 6 carbon atoms; aminoalkyl groups including groups havingone or more N atoms and from 1 to about 8, for example 1, 2, 3, 4, 5 or6, carbon atoms; carbocyclic aryl having 4, 5, 6 or more carbons and oneor more rings, (e.g., phenyl, biphenyl, naphthyl, or the like, each ringeither substituted or unsubstituted aromatic); arylalkyl having 1 to 3separate or fused rings and from 6 to about 18 ring carbon atoms, (e.g.benzyl); arylalkoxy having 1 to 3 separate or fused rings and from 6 toabout 18 ring carbon atoms (e.g. O-benzyl); or a saturated, unsaturated,or aromatic heterocyclic group having 1 to 3 separate or fused ringswith 3 to about 8 members per ring and one or more N, O or S atoms,(e.g., coumarinyl, quinolinyl, isoquinolinyl, quinazolyl, pyridyl,pyrazinyl, pyrimidyl, furanyl, pyrrolyl, thienyl, thiazoyl, triazinyl,oxazolyl, isoxazolyl, imidazolyl, indolyl, benzofuranyl, benzothiazolyl,tetrahydrofuranyl, tetrahyrodropyranyl, piperidinyl, morpholinyl,piperazinyl, and pyrrolidinyl). Such heterocyclic groups may be furthersubstituted, e.g. with hydroxy, alkyl, alkoxy, halogen and amino.

As used herein, “alkyl” is intended to include branched, straight-chain,and cyclic saturated aliphatic hydrocarbon groups. Examples of alkylinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, tert-butyl, n-pentyl, and sec-pentyl. In certainembodiments, alkyl groups are C₁-C₆ alkyl groups or C₁-C₄ alkyl groups.Particular alkyl groups are methyl, ethyl, propyl, butyl, and 3-pentyl.The term “C₁-C₆ alkyl” as used herein means straight-chain, branched, orcyclic C₁-C₆ hydrocarbons which are completely saturated and hybridsthereof such as (cycloalkyl)alkyl. Examples of C₁-C₆ alkyl substituentsinclude methyl (Me), ethyl (Et), propyl (including n-propyl (n-Pr,^(n)Pr), iso-propyl (i-Pr, ^(i)Pr), and cyclopropyl (c-Pr, ^(c)Pr)),butyl (including n-butyl (n-Bu, ^(n)Bu), iso-butyl (i-Bu, ^(i)Bu),sec-butyl (s-Bu, ^(s)Bu), tert-butyl (t-Bu, ^(t)Bu), or cyclobutyl(c-Bu, ^(t)Bu)), and so forth. “Cycloalkyl” is intended to includesaturated ring groups, such as cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl. Cycloalkyl groups typically will have 3 to about 8 ringmembers. In the term “(cycloalkyl)alkyl”, cycloalkyl, and alkyl are asdefined above, and the point of attachment is on the alkyl group. Thisterm encompasses, but is not limited to, cyclopropylmethyl,cyclopentylmethyl, and cyclohexylmethyl.

As used herein, “alkenyl” is intended to include hydrocarbon chains ofeither a straight or branched configuration comprising one or moreunsaturated carbon-carbon bonds, which may occur in any stable pointalong the chain, such as ethenyl and propenyl. Alkenyl groups typicallywill have 2 to about 8 carbon atoms, more typically 2 to about 6 carbonatoms.

As used herein, “alkynyl” is intended to include hydrocarbon chains ofeither a straight or branched configuration comprising one or morecarbon-carbon triple bonds, which may occur in any stable point alongthe chain, such as ethynyl and propynyl. Alkynyl groups typically willhave 2 to about 8 carbon atoms, more typically 2 to about 6 carbonatoms.

As used herein, “haloalkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, substituted with 1 or more halogenatoms. Examples of haloalkyl include, but are not limited to, mono-,di-, or tri-fluoromethyl, mono-, di- or tri-chloromethyl, mono-, di-,tri-, tetra-, or penta-fluoroethyl, and mono-, di-, tri-, tetra-, orpenta-chloroethyl, etc. Typical haloalkyl groups will have 1 to about 8carbon atoms, more typically 1 to about 6 carbon atoms.

As used herein, “alkoxy” represents an alkyl group as defined aboveattached through an oxygen bridge. Examples of alkoxy include, but arenot limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy,2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy,neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy. Alkoxygroups typically have 1 to about 8 carbon atoms, more typically 1 toabout 6 carbon atoms.

As used herein, “haloalkoxy” represents a haloalkyl group as definedabove with the indicate number of carbon atoms attached through anoxygen bridge. Haloalkoxy groups will have 1 to about 8 carbon atoms,more typically 1 to about 6 carbon atoms.

As used herein, “alkylthio” includes those groups having one or morethioether linkages and typically from about 1 to about 8 carbon atoms,more typically 1 to about 6 carbon atoms.

As used herein, the term “alkylsulfinyl” includes these groups havingone or more sulfoxide (SO) linkage groups and typically from 1 to about8 carbon atoms, more typically 1 to about 6 carbon atoms.

As used herein, the term “alkylsulfonyl” includes those groups havingone or more sulfonyl (SO₂) linkage groups and typically from 1 to about8 carbon atoms, more typically 1 to about 6 carbon atoms.

As used herein, the term “alkylamino” includes those groups having oneor more primary, secondary and/or tertiary amine groups and typicallyfrom 1 to about 8 carbon atoms, more typically 1 to about 6 carbonatoms.

As used herein, “Halo” or “halogen” refers to fluoro, chloro, bromo, oriodo; and “counter-ion” is used to represent a small, negatively chargedspecies such as chloride, bromide, hydroxide, acetate, sulfate, and thelike.

As used herein, “carbocyclic group” is intended to mean any stable 3- to7-membered monocyclic or bicyclic or 7- to 13-membered bicyclic ortricyclic group, any of which may be saturated, partially unsaturated,or aromatic. In addition to those exemplified elsewhere herein, examplesof such carbocycles include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl,[3.3.0]bicyclooctanyl, [4.3.0]bicyclononanyl, [4.4.0]bicyclodecanyl,[2.2.2]bicyclooctanyl, fluorenyl, phenyl, naphthyl, indanyl, andtetrahydronaphthyl.

As used herein, the term “aryl” includes groups that contain 1 to 3separate or fused rings and from 6 to about 18 ring atoms, withouthetero atoms as ring members. Example of aryl groups include but are notlimited to phenyl, and naphthyl, including 1-napthyl and 2-naphtyl.

As used herein, “heterocyclic group” is intended to include saturated,partially unsaturated, or unsaturated (aromatic) groups having 1 to 3(possibly fused) rings with 3 to about 8 members per ring at least onering containing an atom selected from N, O or S. The nitrogen and sulfurheteroatoms may optionally be oxidized. The term or “heterocycloalkyl”is used to refer to saturated heterocyclic groups.

A heterocyclic ring may be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure. Theheterocyclic rings described herein may be substituted on carbon or on anitrogen atom if the resulting compound is stable. A nitrogen in theheterocycle may optionally be quarternized.

As used herein, the terra “heteroaryl” is intended to include any stable5- to 7-membered monocyclic or 10- to 14-membered bicyclic heterocyclicaromatic ring system which comprises carbon atoms and from 1 to 4heteroatoms independently selected from the group consisting of N, O andS. In exemplary embodiments, the total number of S and O atoms in thearomatic heterocycle is not more than 2, and typically not more than 1.

Examples of heteroaryl include, but are not limited to, thoseexemplified elsewhere herein and further include acridinyl, azocinyl,benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl,benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl,NH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H.6HA,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isochromanyl,isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl,isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl,oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl; 1,2,5oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl,phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl,phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4thiadiazolyl, thianthrenyl,thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl,thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl,1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl. Exemplary heteroarylgroups include, but are not limited to, pyridinyl, pyrimidinyl, furanyl,thienyl, pyrrolyl, pyrazolyl, pyrrolidinyl, morpholinyl, piperidinyl,piperazinyl, and imidazolyl.

In certain embodiments, Z is tetrazole or CO₂Q. When Z is tetrazole, thetetrazole ring is attached through the carbon atom, as shown below.

In certain embodiments, Q is a protecting group. As used herein, a“protecting group” is a chemical substituent which can be selectivelyremoved by readily available reagents which do not attack theregenerated functional group or other functional groups in the molecule.Suitable protecting groups may be found, for example in Wutz et al.(“Greene's Protective Groups in Organic Synthesis, Fourth Edition,”Wiley-Interscience, 2007). Protecting groups for protection of thecarboxyl group, as described by Wutz et al. (pages 533-643), are used incertain embodiments. In some embodiments, the protecting group isremovable by treatment with acid. Specific examples of protecting groupsinclude but are not limited to, benzyl, p-methoxybenzyl (PMB), tertiarybutyl (^(t)Bu), methoxymethyl (MOM), methoxyethoxymethyl (MEM),methylthiomethyl (MTM), tetrahydropyranyl (THP), tetrahydrofuranyl(THF), benzyloxymethyl (BOM), trimethylsilyl (TMS), triethylsilyl (TES),t-butyldimethylsilyl (TBDMS), and triphenylmethyl (trityl, Tr).

In some embodiments, R⁸ is alkyl, aryl or heteroaryl each of which maybe substituted. In certain embodiments, R⁸ describes the sidechain of anatural or synthetic α-amino acid. Specific examples of R⁸ includehydrogen, methyl (CH₃), isopropyl (CH(CH₃)₂), 2,2-dimethylethyl(CH₂CH(CH3)₂), 2-methylpropyl (CH(CH₃)CH₂CH₃), phenyl, 4-hydroxyphenyl,hydroxymethyl (CH₂OH), carboxymethyl (CH₂CO₂H), thiomethyl (CH₂SH),imidazolylmethyl, indolylmethyl, and so forth.

Certain embodiments include compounds according to formula I where Z isCO₂Q. In other embodiments, Q is hydrogen. In some specific embodiments,Z is CO₂Q and Q is hydrogen.

Certain embodiments include compounds according to formula I, where m is1, 2, 3, or 4.

Other embodiments include compounds according to formula I wherein m is0, 1, 2, 3, 4, 5, or 6; R is a pyridine ring selected from the groupconsisting of

wherein X is fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, chlorine, bromine, a radioisotope of bromine, aradioisotope of astatine, NO₂, NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²),B(OH)₂, —NHNH₂, —NHN═CHR³, —NHNH—CH₂R³. In certain embodiments, n is 1.Each Q is independently selected from hydrogen or a protecting group; Zis tetrazole or CO₂Q; Y is O, S, N(R′), C(O), NR′C(O), C(O)N(R′), OC(O),C(O)O, NR′C(O)NR′, NR′C(S)NR′, NR′S(O)₂, S(CH₂)_(p), NR′(CH₂)_(p),O(CH₂)_(p), OC(O)CHR⁸NHC(O), NHC(O)CHR⁸NHC(O), or a covalent bond;wherein p is 1, 2, or 3, R′ is H or C₁-C₆ alkyl, and R⁸ is alkyl, arylor heteroaryl, each of which may be substituted; R² is C₁-C₆ alkyl; andR³ is alkyl, alkenyl, alkynyl, aryl, or heteroaryl each of which issubstituted by fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, chlorine bromine, a radioisotope of bromine, ora radioisotope of astatine; NO₂, NH₂, N⁺(R²)₃, Sn(R²), Si(R²)₃, Hg(R²),or B(OH)₂. In certain embodiments, R³ is aryl, substituted by fluorine,iodine, a radioisotope of fluorine, a radioisotope of iodine, bromine, aradioisotope of bromine, or a radioisotope of astatine.

Other embodiments include compounds having the structure

wherein m is not 0. R is a pyridine ring selected from the groupconsisting of

wherein X is fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, chlorine, bromine, a radioisotope of bromine, aradioisotope of astatine, NO₂, NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²),B(OH)₂, —NHNH₂, —NHN═CHR³, or —NHNH—CH₂R³. R² is C₁-C₆ alkyl; and R³ isalkyl, alkenyl, alkynyl, aryl, or heteroaryl each of which issubstituted by fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, chlorine bromine, a radioisotope of bromine, ora radioisotope of astatine; NO₂, NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²),or B(OH)₂. In certain embodiments, n is 1. Other specific embodimentsinclude compounds where X is fluorine, iodine, or a radioisotope offluorine, a radioisotope of iodine, chlorine, bromine, a radioisotope ofbromine, or a radioisotope of astatine. In certain embodiments, R³ isaryl, substituted by fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, bromine, a radioisotope of bromine, or aradioisotope of astatine. Specific embodiments include compounds havingthe structure shown above, where Z is CO₂Q, Q is hydrogen, and m is 4.

Compounds according to this embodiment can be prepared, for example,from p-methoxybenzyl (PMB) protected precursor Lys-C(O)-Glu according toScheme 1 shown below.

Other embodiments include compounds having the structure

wherein m is not 0. R is a pyridine ring selected from the groupconsisting of

wherein X is fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, chlorine, bromine, a radioisotope of bromine, aradioisotope of astatine, NO₂, NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²),B(OH)₂, —NHNH₂, —NHN═CHR³, —NHNH—CH₂R³. R² is C₁-C₆ alkyl; and R³ isalkyl, alkenyl, alkynyl, aryl, or heteroaryl each of which issubstituted by fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, chlorine bromine, a radioisotope of bromine, ora radioisotope of astatine; NO₂, NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²),or B(OH)₂. In certain embodiments, n is 1. Other specific embodimentsinclude compounds where X is fluorine, iodine, or a radioisotope offluorine, a radioisotope of iodine, chlorine, bromine, a radioisotope ofbromine, or a radioisotope of astatine. In certain embodiments, R³ isaryl, substituted by fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, bromine, a radioisotope of bromine, or aradioisotope of astatine. Specific embodiments include compounds havingthe structure shown above, where Z is CO₂Q, Q is hydrogen, and m is 1,2, or 3.

Other embodiments include compounds according to formula I wherein R isthe structure below

wherein X′ is selected from the group consisting of —NHNH₂, —NHN═CHR³,—NHNH—CH₂R³. In such embodiments, R³ is alkyl, alkenyl, alkynyl, aryl,or heteroaryl each of which is substituted by fluorine, iodine, aradioisotope of fluorine, a radioisotope of iodine, bromine, aradioisotope of bromine, or a radioisotope of astatine; NO₂, NH₂,N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²), B(OH)₂. R² is C₁-C₆ alkyl; and R³ isalkyl, alkenyl, alkynyl, aryl, or heteroaryl each of which issubstituted by fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, chlorine bromine, a radioisotope of bromine, ora radioisotope of astatine; NO₂, NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²),or B(OH)₂. In certain embodiments, R³ is aryl, substituted by fluorine,iodine, a radioisotope of fluorine, a radioisotope of iodine, bromine, aradioisotope of bromine, or a radioisotope, of astatine. Specificembodiments include compounds where n is 1.

Compounds according to this embodiment can be prepared, for example,from hydrazine substituted phenyl precursors, followed by derivatizationwith an alkyl, alkenyl, alkynyl, aryl, or heteroaryl reagent, each ofwhich is substituted by fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, chlorine bromine, a radioisotope of bromine, ora radioisotope of astatine NO₂, NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²),and B(OH)₂ as illustrated in Scheme 2 below.

Other embodiments include compounds according to formula I wherein m is4, Y is NR′, and R is

wherein G is O, NR′ or a covalent bond, R′ is H or C₁-C₆ alkyl, and p is1, 2, 3, or 4. R⁷ can be selected from NH₂, N═CHR³, and NH—CH₂R³,wherein R³ is alkyl, alkenyl, alkynyl, aryl, or heteroaryl, each ofwhich is substituted by fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, bromine, a radioisotope of bromine, aradioisotope of astatine, NO₂, NH₂, N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²),or B(OH)₂, where R² is C₁-C₆ alkyl. In certain embodiments, R³ is arylsubstituted by fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, chlorine, bromine, a radioisotope of bromine, ora radioisotope of astatine. In certain embodiments, G is O or NR′.

Compounds according to this embodiment can be prepared, for example, byacylation of PMB protected Lys-C(O)-Glu with an acylating agent bearinga free or protected amine, followed by deprotection of the amine, ifnecessary, and derivatization with an alkyl, alkenyl, alkynyl, aryl, orheteroaryl reagent, each of which is substituted by fluorine, iodine, aradioisotope of fluorine, a radioisotope of iodine, chlorine, bromine, aradioisotope of bromine, or a radioisotope of astatine, NO₂, NH₂,N⁺(R²)₃, Sn(R²)₃, Si(R²)₃, Hg(R²), or B(OH)₂, as illustrated by Scheme 3shown below.

Other embodiments include compounds according to any of the embodimentsdiscussed herein, which comprise a radioisotope. Specific exemplaryradioisotopes include ¹⁸F, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁶I, ¹³¹I, ⁷⁵Br, ⁷⁶Br,⁷⁷Br, ⁸⁰Br, ^(80m)Br, ⁸²Br, ⁸³Br and ²¹¹At. Radioisotope containingcompounds of any embodiment of the present invention can be preparedwith sufficient radiolabel to be used in imaging applications. In otherwords, the compounds can be prepared with radioisotope concentrationsgreater than natural abundance, when a particular radioisotope occursnaturally.

Specific examples of compounds according to the previous embodimentsinclude the structures shown below

Other embodiments include pharmaceutically acceptable salts of thecommands described in the previous embodiments.

Compounds according to the invention, in particular, variousradiolabeled compounds, may be used for diagnostic, imaging, ortherapeutic purposes. For example, some compounds, e.g. those labeledwith ¹²⁵I and ¹²³I, are designed for SPECT imaging, while somecompounds, e.g. those labeled with ¹⁸F and ¹²⁴I, are designed for PETimaging, and some radioisotopically labeled compounds may be usedtherapeutically. In general, the suitability of a particularradioisotope for a particular purpose is well understood in the art.Other exemplary embodiments are compounds used us precursors forradiolabeled compounds, in which a substituent may be directly exchangedfor a radioisotope in one or more steps. Unless described otherwise, theterms “converted,” derivatized,” “exchanged,” or “reacted” are intendedto encompass one or more steps. Examples of substituents that may beexchanged for radioisotopes include halogens, NO₂, N⁺(R²)₃, Sn(R²)₃,Si(R²)₃, Hg(R²), and B(OH)₂. Other compounds are precursors which may bechemically reacted with a radioisotopically labeled reagent to produce astable radioisotopically labeled compound. Compounds bearingsubstituents such as halogen, —NH₂, —NHNH₂, Sn(R²)₃, and B(OH)₂, forexample, may be converted into radioisotopically labeled compounds bychemical reactions known to those in the art.

Compounds of the present invention may be made by methods known in theart. For example, the asymmetrical ureas used as precursors may beproduced by the general scheme shown below, where R is the sidechain ofa natural or synthetic amino acid, which bears a group that can befurther derivatized. Specific examples of amino acids include lysine,cysteine, homocysteine, serine, threonine, tyrosine, phenylalanine andsubstituted phenylalanine. Substituted phenylalanine has the structureof phenylalanine where the phenyl sidechain is substituted by, forexample, nitro, amino or halogen.

Protected urea precursors Cys-C(O)-Glu, and Lys-C(O)-Glu (shown below),where Q is p-methoxybenzyl (PMB) are used to synthesize exemplarycompounds. Preparation of precursor Cys-C(O)-Glu is described, forexample by Kozikowski et al. (29), while the preparation of Lys-C(Q)-Gluis described, for example, by Banerjee et al. (19).

Compounds of the present invention may be prepared, for example, byreaction of a protected urea precursor with a reagent substituted with aradioisotope or other substituent which may be converted or derivatizedinto a radioisotope containing compound. A protected urea precursor,such as those described above may be reacted, for example with anactivated benzoate or pyridine carboxylate. The synthesis of both thehalobenzoate and pyridine carboxylate radionuclide-bearing precursorshave been described (20) (21) (22) (23) (25) (37) (38).

Pyridinecarboxylate ¹⁸F precursors, such asn-hydroxysuccinimide-activated pyridine carboxylates, can be prepared,for example, by the scheme shown below.

Other ¹⁸F pyridine precursors may be prepared by methods described byOlberg et al. (J. Labeled Compd. Radiopharm, vol. 52: Supplement 1, p.S160, 2009), shown below.

An ¹⁸F pyridinecarboxylate precursor may be used to prepare compoundsaccording to the present invention, for example, according to the schemeshown below.

Similarly, a precursor may be prepared, which can then be converted intoan ¹⁸F-substituted compound. For example, compounds may be preparedaccording to the scheme below.

Other compounds, may be produced from a suitable protected precursor,such as those discussed above, by reaction with bromomethyl pyridinecompounds bearing radioisotope substituents, or substituents which maybe converted to radioisotopes or derivatized with radioisotopecontaining compounds. For example, the scheme below shows preparation ofan exemplary compound from PMB protected precursor Cys-C(O)-Glu.

Bromomethyl pyridine compounds, such as ¹⁸F, NO₂ or N(CH₃)₃ ⁺substituted bromomethyl pyridine, suitable for preparation of compoundsaccording to the present invention can be prepared, for example,according to the scheme shown below.

Other compounds can be prepared, for example, from hydrazine (—NHNH₂)substituted pyridine precursors, followed by derivatization with analkyl, alkenyl, alkynyl, aryl, or heteroaryl reagent each of which issubstituted by fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, chlorine, bromine, a radioisotope of bromine, ora radioisotope of astatine, NO₂, NH₂, N⁺(R²)₃, Sn(R²), Si(R²)₃, Hg(R²),and B(OH)₂. For example, an aldehyde reagent may be reacted with thehydrazine substituent, as illustrated in Scheme 4 shown below. Theresulting imine may also be reduced, for example, by sodiumcyanoborohydride or other reducing agent, to produce a reduced compound

Other embodiments include compounds having the formula

wherein R⁵ is hydrogen or p-methoxybenzyl; R⁴ is selected from the groupconsisting of hydrogen,

wherein A is fluorine, iodine, a radioisotope of fluorine, aradioisotope of iodine, bromine, a radioisotope of bromine, aradioisotope of astatine, Sn(R²)₃, Si(R²)₃, or HgCl. Further embodimentsinclude compounds according to the structure above, which comprise aradioisotope. One specific embodiment includes the compound having theformula shown below, also known as PMB protected Lys-C(O)-Glu.

wherein PMB is p-methoxybenzyl. Another specific embodiment includes thecompound 2-[3-(5-amino-1-carboxy-pentyl)-ureido]-pentanedioic acid, alsoknown as Lys-C(O)-Glu. Other exemplary embodiments include compoundsshown below.

Exemplary radioisotope containing compounds include the compounds shownbelow.

Other embodiments of the inventions include methods of imaging one ormore cells, organs or tissues comprising exposing cells to oradministering to a subject an effective amount of a compound with anisotopic label suitable for imaging. In some embodiments, the one ormore organs or tissues include prostate tissue, kidney tissue, braintissue, vascular tissue or tumor tissue.

In another embodiment, the imaging method is suitable for imagingstudies of PSMA inhibitors, for example, by studying competitive bindingof non-radiolabeled inhibitors. In still another embodiment, the imagingmethod is suitable for imaging of cancer, tumor or neoplasm. In afurther embodiment, the cancer is selected from eye or ocular cancer,rectal cancer, colon cancer, cervical cancer, prostate cancer, breastcancer and bladder cancer, oral cancer, benign and malignant tumors,stomach cancer, liver cancer, pancreatic cancer, lung cancer, corpusuteri, ovary cancer, prostate cancer, testicular cancer, renal cancer,brain cancer (e.g., gliomas), throat cancer, skin melanoma, acutelymphocytic leukemia, acute myelogenous leukemia, Ewing's Sarcoma,Kaposi's Sarcoma, basal cell carinoma and squamous cell carcinoma, smallcell lung, cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma,hemangioendothelioma, Wilms Tumor, neuroblastoma, mouth/pharynx cancer,esophageal cancer, larynx cancer, lymphoma, neurofibromatosis, tuberoussclerosis, hemangiomas, and lymphangiogenesis.

The imaging methods of the invention are suitable for imaging anyphysiological process or feature in which PSMA is involved. Typically,imaging methods are suitable for identification of areas of tissues ortarget which express high concentrations of PSMA. Typical applicationsinclude imaging glutamateric neurotransmission, presynapticglutamatergic neurotransmission, malignant tumors or cancers thatexpress PSMA, prostate cancer (including metastasized prostate cancer),and angiogenesis. Essentially all solid tumors express PSMA in theneovasculture. Therefore, methods of the present invention can be usedto image nearly all solid tumors including lung, renal cell,glioblastoma, pancreas, bladder, sarcoma, melanoma, breast, colon, germcell, pheochromocytoma, esophageal and stomach. Also, certain benignlesions and tissues including endometrium, schwannoma and Barrett'sesophagus can be imaged according to the present invention.

The methods of imaging angiogenesis provided by the present inventionare suitable for use in imaging a variety of diseases and disorders inwhich angiogenesis takes place. Illustrative, non-limiting, examplesinclude tumors, collagen vascular disease, cancer, stroke, vascularmalformations, retinopathy. Methods of imaging angiogenesis provided bythe present invention, are also suitable for use in diagnosis andobservation of normal tissue development.

PSMA is frequently expressed in endothelial cells of capillary vesselsin peritumoral and endotumoral areas of various malignancies such thatcompounds of the invention and methods of imaging using same aresuitable for imaging such malignancies.

In certain embodiments, the radiolabeled compound is stable in vivo.

In certain embodiments, the radiolabeled compound is detected bypositron emission tomography (PET) or single photon emission computedtomography (SPECT).

In one embodiment, the invention provides a method wherein the subjectis a human, rat, mouse, cat, dog, home, sheep, cow, monkey, avian, oramphibian. In another embodiment, the cell is in vivo or in vitro.Typical subjects to which compounds of the invention may be administeredwill be mammals, particularly primates, especially humans. Forveterinary applications, a wide variety of subjects will be suitable,e.g. livestock such as cattle, sheep, goats, cows, swine, and the like;poultry such as chickens, ducks, geese, turkeys, and the like; anddomesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects including rodents (e.g. mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like.Additionally, for in vitro applications, such as in vitro diagnostic andresearch applications, body fluids and cell samples of the abovesubjects will be suitable for use such as mammalian, particularlyprimate such as human, blood, urine or tissue samples, or blood urine ortissue samples of the animals mentioned for veterinary applications.

In certain methods of the invention the compounds of the invention areexcreted from tissues of the body quickly to prevent prolonged exposureto the radiation of the radiolabeled compound administered to thepatient. Typically compounds of the invention are eliminated from thebody in less than about 24 hours. More typically, compounds of theinvention are eliminated from the body in less than about 16 hours, 12hours, 8 hours, 6 hours, 4 hours, 2 hours, 90 minutes, or 60 minutes.Exemplary compounds are eliminated in between about 60 minutes and about120 minutes.

In some embodiments, the compounds bind strongly to the PSMA protein,for instance by incorporating structural features which reside in anaccessory binding site. For example, in compound 3, the 4-iodobenzylgroup resides in a hydrophobic pocket accessory to the S1 binding site(39).

In certain embodiments, compounds of the invention are stable in vivosuch that substantially all, e.g., more than about 50%, 60%, 70%, 80%,or 90% of the injected compound is not metabolized by the body prior toexcretion. Typical subjects to which compounds of the invention may beadministered will be mammals, particularly primates, especially humans.For veterinary applications, a wide variety of subjects will besuitable, e.g. livestock such as cattle, sheep, goats, cows, swine andthe like; poultry such as chickens, ducks, geese, turkeys, and the like;and domesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects including rodents (e.g. mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the life.Additionally, for in vitro applications, such as in vitro diagnostic andresearch applications, body fluids and cell samples of the abovesubjects will be suitable for use such as mammalian, particularlyprimate such as human, blood, urine or tissue samples, or blood urine ortissue samples of the animals mentioned for veterinary applications.

Other embodiments of the indention provide methods of treating tumorscomprising administering to a subject a therapeutically effective amountof a compound according to the present invention comprising atherapeutically effective radioisotope. In certain embodiments, thetumor cells may express PSMA, such as prostate tumor cells ormetastasized prostate tumor cells. In other embodiments, a tumor may betreated by targeting adjacent or nearby cells which express PSMA. Forexample, vascular cells undergoing angiogenesis associated with a tumormay be targeted. Essentially all solid tumors express PSMA in theneovasculature. Therefore, methods of the present invention can be usedto treat nearly all solid tumors including lung, renal cell,glioblastoma, pancreas, bladder, sarcoma, melanoma, breast, colon, germcell, pheochromocytoma, esophageal and stomach. Also, certain benignlesions and tissues including endometrium, schwannoma and Barrett'sesophagus can be treated according to the present invention. Examples oftherapeutically radioisotopes include ¹³¹I and ²¹¹At.

Other embodiments provide kits comprising a compound according to theinvention. In such embodiments, the kit provides packaged pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and acompound of the invention. In certain embodiments the packagedpharmaceutical composition will comprise the reaction precursorsnecessary to generate the compound of the invention upon combinationwith a radiolabeled precursor. Other packaged pharmaceuticalcompositions provided by the present invention further comprise indiciacomprising at least one of: instructions for preparing compoundsaccording to the invention from supplied precursors, instructions forusing the composition to image cells or tissues expressing PSMA, orinstructions for using the composition to image glutamatergicneurotransmission in a patient suffering from a stress-related disorder,or instructions for using the composition to image prostate cancer.

In certain embodiments, a kit according to the invention contains fromabout 1 to about 30 mCi of the radionuclide-labeled imaging agentdescribed above, in combination with a pharmaceutically acceptablecarrier. The imaging agent and carrier may be provided in solution or inlyophilized form. When the imaging agent and carrier of the kit are inlyophilized form, the kit may optionally contain a sterile andphysiologically acceptable reconstitution medium such as water, saline,buffered saline, and the like. The kit may provide a compound of theinvention in solution or in lyophilized form, and these components ofthe kit of the invention may optionally contain stabilizers such asNaCl, silicate, phosphate buffers, ascorbic acid, gentisic acid, and thelike. Additional stabilization of kit components may be provided in thisembodiment, for example, by providing the reducing agent in anoxidation-resistant form. Determination and optimization of suchstabilizers and stabilization methods are well within the level of skillin the art.

In certain embodiments, a kit provides a non-radiolabeled precursor tobe combined with a radiolabeled reagent on-site. Examples of radioactivereagents include Na[¹²⁵I], Na[¹³¹I], Na[¹²³I], Na[¹²⁴I], K[¹⁸F],Na[⁷⁶Br], Na[²¹¹At]. Other radiolabeled reagents include activatedradiolabeled benzoyl compounds, radiolabeled pyridine carboxylates,radiolabeled bromomethyl pyridine compounds, and radiolabeled aldehydesdiscussed previously.

Imaging agents of the invention may be used in accordance with themethods of the invention by one of skill in the art. Images can begenerated by virtue of differences in the spatial distribution of theimaging agents which accumulate at a site when contacted with PSMA. Thespatial distribution may be measured using any means suitable for theparticular label for example, a gamma camera, a PET apparatus, a SPECTapparatus, and the like. The extent of accumulation of the imaging agentmay be quantified using known methods for quantifying radioactiveemissions. A particularly useful imaging approach employs more than oneimaging agent to perform simultaneous studies.

In general, a detectably effective amount of the imaging agent of theinvention is administered to a subject. In accordance with theinvention, “a detectably elective amount” of the imaging agent of theinvention is defined as an amount sufficient to yield an acceptableimage using equipment which is available for clinical use. A detectablyeffective amount of the imaging agent of the invention may beadministered in more than one injection. The detectably effective amountof the imaging agent of the invention can vary according to factors suchas the degree of susceptibility of the individual, the age, sex, andweight of the individual, idiosyncratic responses of the individual, andthe dosimetry. Detectably effective amounts of the imaging agent of theinvention can also vary according to instrument and film-relatedfactors. Optimization of such factors is well within the level of skillin the art. The amount of imaging agent used for diagnostic purposes andthe duration of the imaging study will depend upon the radionuclide usedto label the agent, the body mass of the patient, the nature andseverity of the condition being treated, the nature of therapeutictreatments which the patient has undergone, and on the idiosyncraticresponses of the patient. Ultimately, the attending physician willdecide the amount of imaging agent to administer to each individualpatient and the duration of the imaging study.

A “pharmaceutically acceptable carrier” refers to a biocompatiblesolution, having due regard to sterility, p[Eta], isotonicity,stability, and the like and can include any and all solvents, diluents(including sterile saline, Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,Lactated Singer's Injection and other aqueous buffer solutions),dispersion media, coatings, antibacterial and antifungal agents,isotonic agents, and the like. The pharmaceutically acceptable carriermay also contain stabilizers, preservatives, antioxidants, or otheradditives, which are well known to one of skill in the art, or othervehicle as known in the art.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking non-toxic acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid safe of basic residues such as amines; alkali ororganic salts of acidic residues such as carboxylic acids; and the like.The pharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,conventional non-toxic acid salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, malefic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, mesylic, sulfanilic, 2-acetoxybenzoic,fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic,isethionic, HOOC—(CH₂)_(n)—COOH where n is 0-4, and the like. Thepharmaceutically acceptable salts of the present invention can besynthesized from a parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting free acid forms of these compounds with astoichiometric amount of the appropriate base (such as Na, Ca, Mg, or Khydroxide, carbonate, bicarbonate, or the like), or by reacting freebase forms of these compounds with a stoichiometric amount of theappropriate acid. Such reactions are typically carried out in water orin an organic solvent, or in a mixture of the two. Generally,non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, oracetonitrile are used, where practicable. Lists of additional suitablesalts may be found, e.g., in Remington's Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).

The contents of all cited references (including literature references,issued patents, published patent applications) as cited throughout thisapplication are hereby expressly incorporated by reference. Theinvention and the manner and process of making and using it, aredescribed in such full, clear, concise and exact terms as to enable anyperson skilled in the art to which it pertains, to make and use thesame.

It is to be understood that the foregoing describes exemplaryembodiments of the present invention and that modifications may be madetherein without departing from the spirit or scope of the presentinvention as set forth in the appended claims.

EXAMPLES

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The practice ofthe present invention will employ, unless otherwise indicated,conventional techniques, which are within the skill of the art. Suchtechniques are explained fully in the literature.

Synthesis

General Procedures.

All reagents and solvents were purchased from either Sigma-Aldrich(Milwaukee, Wis.) or Fisher Scientific (Pittsburgh, Pa.). The tosylatesalt of PMB-protected Lys-C(O)-Glu (compound 1) was prepared accordingto a reported procedure (19). ¹H NMR spectra were obtained on a VarianMercury 400 mHz or a Bruker Avance 400 mHz Spectrometer. ESI massspectra were obtained on an API 15OEX™ or a Bruker Esquire 3000 plussystem. High-resolution mass spectra (HRMS) were performed on a JEOLJMS-AX505HA mass spectrometer in the Mass Spectrometry Facility at theUniversity of Notre Dame. HPLC purification of reference compounds wasperformed on a Waters 625 LC system with a Waters 490E multiwavelengthUV/Vis detector (Milford, Mass.).

[¹²⁵I]NaI was purchased from MP Biomedicals (Costa Mesa, Calif.).[¹⁸F]Fluoride was produced by 18 MeV proton bombardment of a highpressure [¹⁸O]H₂O target using a General Electric PETtrace biomedicalcyclotron (Milwaukee, Wis.). Solid-phase extraction cartridges (C₁₈plus, Sep-Pak) were purchased from Waters Associates. Radioactivity wasmeasured in a Capintec CRC-10R dose calibrator (Ramsey, N.J.). Thespecific radioactivity was calculated as the radioactivity eluting atthe retention time of product during the semi-preparative HPLCpurification divided by the mass corresponding to the area under thecurve of the UV absorption.

Example 12-{3-[5-(4-Iodo-benzoylamino)-1-(4-methoxy-benzyloxycarbonyl)-pentyl]-ureido}-pentanedioicacid bis-(4-methoxy-benzyl) ester (2)

To a solution of 1 (0.126 g, 0.148 mmol) in CH₂Cl₂ (4 ml) was addedtriethylamine (0.1 mL, 0.712 mmol), followed byN-hydroxysuccinimidyl-4-iodobenzoate (24) (0.073 g, 0.212 mmol). Afterstirring for 2 h at room temperature, the solvent was evaporated on arotary evaporator. The crude material was purified on a silica columnusing methanol/methylene chloride (5:95) to afford 0.127 g (94%) of 2.¹H NMR (400 MHz, CDCl₃) δ7.69 (d, J=8.8 Hz, 2H), 7.49 (d, J=8.8 Hz, 2H),7.17-7.26 (m, 6H), 6.77-6.86 (m, 7H), 5.37-5.46 (m, 2H), 4.93-5.09 (m,6H), 4.32-4.40 (m, 2H), 3.76-3.77 (m, 9H), 3.30-3.33 (m, 2H), 2.30-2.36(m, 2H), 2.07-2.12 (m, 1H), 1.84-1.92 (m, 1H), 1.70-1.79 (m, 1H),1.49-1.57 (m, 3H), 1.25-1.33 (m, 2H). ESI-Mass calcd for C₄₃H₄₈IN₃O₁₁Na[M+Na]⁺ 932.2, found 932.7.

Example 22-{3-[1-Carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido}-pentanedioicacid (3)

A solution of 3% anisole in TFA (15 mL) was added to 2 (0.117 g, 0.129mmol) at 0° C. The mixture was stirred at room temperature for 30 minthen concentrated on a rotary evaporator. The crude material waspurified by HPLC (Econosil C18 10μ, 250×10 mm, H₂O/CH₃CN/TFA(70/30/0.1), 4 mL/min, 3 eluting at 11 min) to afford 0.040 g (57%) of3. ¹H NMR (400 MHz, D₂O:CD₃CN=1:1 (v/v) δ7.79 (d, J=8.0 Hz, 2H), 7.46(d, J=8.0 Hz, 2H), 4.08-4.16 (m, 2H), 3.26 (m, 2H), 2.35 (m, 2H),2.00-2.03 (m, 1H), 1.72-1.84 (m, 2H), 1.52-1.62 (m, 3H), 1.34-1.36 (m,2H). ESI-Mass calcd for C₁₉H₂₄IN₃O₈Na [M+Na]⁺ 572.1, found 572.0,FAB-HRMS calcd for C₁₉H₂₅IN₃O₈ [M+H]⁺ 550.0686, found 550.0648.

Example 32-{3-[1-(4-Methoxy-benzyloxycarbonyl)-5-(4-tributylstannyl-benzoylamino)-pentyl]-ureido}-pentanedioicacid bis-(4-methoxy-benzyl) ester (4)

To a solution of 1 (0.120 g, 0.148 mmol) in CH₂Cl₂ (6 mL) was addedtriethylamine (0.1 ml, 0.712 mmol), followed byN-hydroxysuccinimidyl-4-tributylstannylbenzoate (24) (0.075 g, 0.147mmol). After stirring for 2 h at room temperature, the reaction mixturewas condensed on a rotary evaporator. The crude material was purified ona silica column using methanol/methylene chloride (5:95) to afford 0.130g (86%) of 4. ¹H NMR (400 MHz, CDCl₃) δ7.68 (d, J=8.4 Hz, 2H), 7.49 (d,J=7.6 Hz, 2H), 7.18-7.24 (m, 6H), 6.80-6.85 (m, 6H), 6.47 (m, 1H),5.44-5.47 (m, 2H), 4.95-5.09 (m, 6H), 4.41-4.45 (m, 2H), 3.76-3.77 (m,9H), 3.32-3.38 (m, 2H), 2.35-2.37 (m, 2H), 2.08-2.16 (m, 1H), 1.90-1.94(m, 1H), 1.70-1.79 (m, 1H), 1.45-1.64 (m, 9H), 1.24-1.30 (m, 8H),1.01-1.06 (m, 6H), 0.85-0.87 (m, 9H). ESI-Mass calcd for C₅₅H₇₅N₃O₁₁SnNa[M+Na]⁺ 1096.4, found 1096.7.

Example 42-{3-[5-(4-Fluoro-benzoylamino)-1-(4-methoxy-benzyloxycarbonyl)-pentyl]-ureido}-pentanedioicacid bis-(4-methoxy-benzyl) ester (5)

To a solution of 1 (0.120 g, 0.164 mmol) in CH₂Cl₂ (4 ml) was addedtriethylamine (0.1 mL, 0.712 mmol), followed byN-hydroxysuccinimidyl-4-fluorobenzoate (22) (0.043 g, 0.181 mmol). Afterstirring for 2 h at room temperature, the solvent was evaporated on arotary evaporator. The crude material was purified by on a silica columnusing methanol/methylene chloride (5:95) to afford 0.120 g (91%) of 5.¹H NMR (400 MHz, CDCl₃) δ7.78 (m, 2H), 7.16-7.24 (m, 6H), 7.01 (m, 2H),6.80-6.85 (m, 7H), 5.51-5.64 (m, 2H), 4.93-5.09 (m, 6H), 4.34-4.40 (m,2H), 3.75-3.77 (m, 9H), 3.28-3.34 (m, 2H), 2.26-2.38 (m, 2H), 2.04-2.15(m, 1H), 1.82-1.91 (m, 1H), 1.68-1.74 (m, 1H), 1.44-1.57 (m, 3H),1.25-1.33 (m, 2H). ESI-Mass calcd for C₄₃H₄₈FN₃O₁₁Na [M+Na]⁺ 824.3,found 824.7.

Example 52-{3-[1-Carboxy-5-(4-fluoro-benzylamino)-pentyl]-ureido}-pentanedioicacid (6)

A solution of 3% anisole in TFA (15 mL) was added to 5 (0.081 g, 0.1mmol) at 0° C. The mixture was stirred at room temperature for 20 minthen concentrated on a rotary evaporator. The crude material waspurified by HPLC (Econosil C18 10 μm, 250×10 mm, H₂O/CH₃CN/TFA(75/25/0.1), 4 mL/min, with purified 6 eluting at about 9 min) to afford0.035 g (79%) of 6. ¹H NMR (400 MHz, D₂O) δ7.66-7.69 (m, 2H), 7.11-7.16(m, 2H), 4.12-4.19 (m, 2H), 3.28-3.31 (m, 2H), 2.39-2.43 (m, 2H),2.07-2.09 (m, 1H), 1.79-1.90 (m, 2H), 1.55-1.69 (m, 3H), 1.39-1.40 (m,2H). ESI-Mass calcd for C₁₉H₂₄FN₃O₈Na [M+Na]⁺ 464.1, found 464.4.FAB-HRMS calcd for C₁₉H₂₅FN₃O₈ [M+H]⁺ 442.1626, found 442.1646.

Example 62-(3-{1-(4-methoxy-benzyloxycarbonyl)-5[(5-tributylstannanyl-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioicacid bis-(4-methoxy-benzyl) ester (7)

To a solution of 1 (0.120 g, 0.148 mmol) in CH₂Cl₂ (2 mL) was addedtriethylamine (0.1 mL, 0.712 mmol), followed byN-hydroxysuccinimidyl-5-(tri-n-butylstannyl)-3-pyridinecarboxylate (25)(0.075 g, 0.147 mmol). After stirring for 30 min at room temperature,the crude material was purified on a silica column usingmethanol/methylene chloride (5:95) to afford 0.115 g (76%) of 7. ¹H NMR(400 MHz, CDCl₃) δ8.85 (s, 1H), 8.65 (s, 1H), 8.19 (s, 1H), 7.19-7.24(m, 6H), 6.81-6.85 (m, 6H), 6.65 (m, 1H), 5.32-5.35 (m, 1H), 5.22-5.25(m, 1H), 4.96-5.10 (m, 6H), 4.40-4.47 (m, 2H), 3.70-3.77 (m, 9H), 3.34(m, 2H), 2.35-2.39 (m, 2H), 2.10-2.15 (m, 1H), 1.90-1.94 (m, 1H),1.72-1.79 (m, 1H), 1.46-1.59 (m, 9H), 1.27-1.36 (m, 8H), 1.02-1.25 (m,6H), 0.84-0.87 (m, 9H). ESI-Mass calcd for C₅₄H₇₅IN₄O₁₁Sn [M+H]⁺ 1075.4,found 1075.5.

Example 72-(3-{1-carboxy-5-[(5-iodo-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioicacid (8)

To a solution of 7 (0.025 g, 0.023 mmol) in 2 ml methanol was added0.020 mL acetic acid and sodium iodide (0.017 g, 0.113 mmol), followedby N-chlorosuccinimide (0.025 g, 0.187 mmol). After 20 min at roomtemperature, the solvent was removed under a stream of nitrogen. Asolution of TFA in CH₂Cl₂ (1:1, 2 mL) was then added to the residue.After 1 h at room temperature, 8 (0.008 g, 62%) was isolated by HPLC(Econosphere C18 10μ, 250×10 mm, H₂O/CH₃CN/TFA (85/15/0.1), 4 mL/min,product peak eluting at 10 min). ¹H NMR (400 MHz, D₂O) δ9.00-9.15 (m,3H), 4.18-4.24 (m, 2H), 3.40-3.41 (m, 2H), 2.45-2.49 (m, 2H), 2.12-2.13(m, 1H), 1.85-1.97 (m, 2H), 1.64-1.73 (m, 3H), 1.44 (m, 2H). ESI-Masscalcd for C₁₈H₂₄IN₄O₈ [M+H]⁺ 551.1, found 551.0. FAB-HRMS calcd forC₁₈H₂₄IN₄O₈ [M+H]⁺ 551.0639, found 551.0607.

Example 82-{3-[1-carboxy-5-(4-[¹²⁵I]iodo-benzoylamino)-pentyl]-ureido}-pentanedioicacid ([¹²⁵I]3)

[¹²⁵I]3 was prepared via iododestannylation of the correspondingtri-n-butylstannyl precursor 4 followed by deprotection. To a solutionof 4 (1 mg, 0.932 μmol) in 0.1 mL methanol was added 0.001 mL aceticacid and [¹²⁵I]NaI, followed by N-chlorosuccinimide (0.25 mg, 0.187μmol) in 0.025 mL of methanol. After stirring at room temperature for 20min, the solvent was removed under a stream of N₂. A solution of 3%anisole in TFA (0.1 mL) was then added to the residue. After 5 min atroom temperature, [¹²⁵I]3 was isolated by HPLC (Econosil C18 10μ,250×4.6 mm, H₂O/CH₃CN/TFA (72/28/0.1), 1 mL/min). Reverse phaseradio-HPLC purification of [¹²⁵I]3 was performed using a Waters 510pump, Waters 490E variable wavelength UV/Vis detector at 254 nm and aBioscan Flow Count PMT radioactivity detector (Washington, D.C.). Theyields of this reaction were 65-80% (three separate determinations).Specific radioactivity was always >700 Ci/mmol (25.9 GBq/μmol).

Example 9 N-Hydroxysuccinimidyl-4-[¹⁸F]fluorobenzoate [¹⁸F]SFB

N-hydroxysuccinimidyl-4-[¹⁸F]iodobenzoate ([¹⁸F]SFB) was prepared by aliterature procedure (23) and purified by HPLC (Econosphere C18 10μ,250×10 mm, H₂O/CH₃CN/TFA (75/25/0.1), 5 mL/min, product peak eluting at19 min).

2{3-[1-carboxy-5-(4-[¹⁸F]fluoro-benzylamino)-pentyl]-ureido}-pentanedioicacid ([¹⁸F]6)

In a vial containing 2 mg of 1 and 0.002 ml of Et₃N was added [¹⁸F]SFBin CH₂Cl₂. The reaction mixture was heated at 45° C. for 20 min, andthen the solvent was removed under a stream of nitrogen. 0.1 mL of 3%anisole/TFA was then added and the reaction mixture was heated at 45° C.for 5 min. The final product was obtained after HPLC purification(Econosphere C18 10μ, 250×10 mm, H₂O/CH₃CN/TFA [80/20/0.1], 4 mL/min).The decay-corrected yields of [¹⁸F]6 were 30-35%, based on starting[¹⁸F]fluoride (three separate determinations). The mean synthesis timewas 180 min from the time of addition of [¹⁸F]fluoride. Starting from 40mCi [¹⁸F]fluoride, the specific radioactivity of [¹⁸F]6 was found to be250-300 Ci/mmol (9.1-11.1 GBq/μmol).

Example 102-(3-{1-carboxy-5-[(5-[¹²⁵I]iodo-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioicacid ([¹²⁵I]8)

[¹²⁵I]8 was prepared using iododestannylation of the correspondingtri-n-butylstannyl precursor 7 followed by deprotection. To a solutionof 7 (0.05 mg, 0.047 μmol) in 0.05 mL of methanol was added 0.002 mLacetic acid, [¹²⁵I]NaI, followed by N-chlorosuccinimide (0.1 mg, 0.749μmol) in 0.010 mL of methanol. After 20 min at room temperature, thesolvent was removed under a steam of nitrogen. A solution of 3% anisolein TFA (0.1 mL) was then added to the residue. After 5 min at roomtemperature, [¹²⁵I]8 was isolated by HPLC (Econosil C18 10μ, 250×4.6 mm,H₂O/CH₃CN/TFA [85/15/0.1], 1 mL/min). Reverse phase radio-HPLCpurification of [¹²⁵I]8 was performed using a Waters 510 pump, Waters490E variable wavelength UV/Vis detector at 354 nm and a Bioscan FlowCount PMT radioactivity detector (Washington D.C.). The yields of thisreaction were 59-75% (three separate determinations). Specificradioactivity was >2,000 Ci/mmol (74.0 GBq/μmol) in each case.

Example 11 Synthesis of2-(3-{1-carboxy-5-[2-(4-fluoro-benzylideneaminooxy)-acetylamino]-pentyl}-ureido)-pentanedioicacid (9)

To a solution of compound 1 (0.062 g, 0.073 mmol) in CH₂Cl₂ (2 mL) wasadded triethylamine (0.045 mL, 0.320 mmol), followed byN-tert-butyloxycarbonyl-O-(carboxymethyl)hydroxyaminehydroxysuccinimidyl ester (0.025 g, 0.087 mmol), Bioconjugate Chemistry1993, 4, 515-20). After stirring for 30 min at room temperature, thesolvent was evaporated. The crude material was purified by a silicacolumn using methanol/methylene chloride (5:95) to afford 0.055 g (89%)of compound 10. ¹H NMR (400 MHz, CDCl₃) δ7.98 (bs, 1H), 7.91 (s, 1H),7.25-7.27 (m, 6H), 6.85-6.88 (m, 6H), 5.56-5.63 (m, 2H), 5.01-5.11 (m,6H), 4.47-4.53 (m, 1H), 4.27-4.38 (m, 3H), 3.79 (m, 9H), 3.30-3.38 (m,1H), 3.15-3.21 (m, 1H), 2.36-2.41 (m, 2H), 2.10-2.15 (m, 1H), 1.89-1.95(m, 1H), 1.74-1.81 (m, 1H), 1.23-1.61 (m, 14H). ESI-Mass calcd forC₄₃H₅₇N₄O₁₄ [M+H]⁺ 853.4, found 853.5.

A solution of 3% anisole in TFA (1 mL) was added to compound 10 (0.031g, 0.036 mmol). The mixture was stirred at room temperature for 5 min,then concentrated on a rotary evaporator. The crude material waspurified by HPLC (Econosil C18 10μ, 250×10 mm, H₂O/CH₃CN/TFA(90/10/0.1), 4 ML/min) to afford 0.009 g (49%) of compound 11. ¹H NMR(400 MHz, D₂O) δ4.68 (s, 2H), 4.28-4.35 (m, 2H), 3.34 (m, 2H), 2.58 (m,2H), 2.24 (m, 1H), 1.78-2.13 (m, 3H), 1.62 (m, 2H), 1.49 (m, 2H).ESI-Mass calcd for C₁₄H₂₅N₄O₉ [M]⁺ 393.2, found 393.3.

To a solution of compound 11 (0.005 g, 0.010 mmol) in methanol (0.3 mL)was added triethylamine (0.0075 ml, 0.05 mmol), followed by4-fluorobenzaldehyde (0.0017 ml, 0.016 mmol). After 30 min at roomtemperature, the reaction mixture was purified by HPLC (Econosil C1810μ, 250×10 mm, H₂O/CH₃CN/TFA (75/25/0.1) and compound 9 (0.002 g, 41%)was obtained. ¹H NMR (400 MHz, D₂O:CD₃CN=1:1) δ8.26 (s, 1H), 7.56-7.80(m, 2H), 7.10-7.14 (m, 2H), 4.53 (s, 2H), 4.13-4.17 (m, 1H), 3.96-4.00(m, 1H), 3.16 (m, 2H), 2.37 (m, 2H), 2.10-2.16 (m, 1H), 1.80-1.88 (m,2H), 1.65 (m, 1H), 1.54 (m, 1H), 1.42 (m, 1H), 1.28 (m, 1H). ESI-Masscalcd for C₂₁H₂₇F₄O₉Na [M+Na]⁺ 521.2, found 521.3, FAB-HRMS calcd forC₂₁H₂₈FN₄O₉ [M+H]⁺ 499.1840, found 499.1869.

Example 12 Synthesis of2-(3-{1-carboxy-5-[2-(4-[¹⁸F]fluoro-benzylideneaminooxy)-acetylamino]-pentyl}-ureido)-pentanedioicacid ([¹⁸F]9)

4-[¹⁸F]fluorobenzaldehyde was synthesized by using literature procedure(Nuclear Medicine and Biology 19 (1992) 275-281) and purified by HPLC(H₂O/CH₃CN/TFA 70/30/0.1). The HPLC eluent of 4-[¹⁸F]fluorobenzaldehydewas diluted with H₂O, passed through a C18 Sep Pak, eluted with 2 mL ofmethanol. To a solution of compound 11 (1 mg) in methanol (0.05 mL) wasadded [¹⁸F]4-fluorobenzaldehyde in 2 mL methanol. After stirred 30 minat room temperature, the reaction mixture was purified by HPLC to givecompound [¹⁸F]9. The radiochemical yield (decay corrected and based onstarting [¹⁸F]fluoride, n=2) was 6-9%. The specific activity of finalcompound was found to be 350-1300 mCi/μmol.

Example 13 Synthesis of2-[3-(1-carboxy-5-{4-[N′-(4-fluoro-benzylidene)-hydrazino]-benzoylamino}-pentyl)-ureido]pentanedioicacid (12)

To a solution of compound 1 (0.030 g, 0.035 mmol) in CH₂Cl₂ (2 mL) wasadded triethylamine (0.020 mL, 0.142 mmol), followed by succinimidyl4-[2-(tert-butoxycarbonyl)hydrazino benzoate (0.020 g, 0.057 mmol,Bioconjugate Chem. 1991, 2, 333-336) in DMF (0.2 mL). After stirred for1 hour at room temperature, the solvent was evaporated. The crudematerial was purified by a silica column using methanol/methylenechloride (5:95) to afford 0.025 g (78%) of compound 13. ¹H NMR (400 MHz,CDCl₃) δ7.63 (d, J=8.0 Hz, 2H), 7.19-7.23 (m, 6H), 6.81-6.85 (m, 6H),6.68 (d, J=8.8 Hz, 2H), 6.61 (s, 2H), 6.15 (bs, 1H) 5.68 (m, 2H),4.95-5.07 (m, 6H), 4.34-4.45 (m, 2H), 3.74 (m, 9H), 3.25 (m, 2H), 2.31(m, 2H), 2.10 (m, 1H), 184 (m, 1H), 1.19-1.74 (m, 14H), ESI-Mass calcdfor C₄₅H₅₉N₅O₁₃Na [M+Na]⁺ 936.4, found 935.9.

A solution of TFA/CH₂Cl₂ 1:1 (2 mL) was added to compound 13 (0.025 g,0.027 mmol). The mixture was stirred at room temperature for 1 hour,then concentrated on a rotary evaporator. The crude material waspurified by HPLC (Econosphere C18 10μ, 250×10 mm, H₂O/CH₃CN/TFA(92/8/0.1), 4 mL/min) to afford 0.010 g (64%) of compound 14. ¹H NMR(400 MHz, D₂O) δ7.72 (d, J=8.8 Hz, 2H), 7.02 (d, J=8.8 Hz, 2H),4.15-4.23 (m, 2H), 3.35 (m, 2H), 2.46 (m, 2H), 2.10-2.17 (m, 1H),1.80-1.95 (m, 2H), 1.59-1.74 (m, 3H), 1.45 (m, 2H), ESI-Mass calcd forC₁₉H₂₈N₅O₈ [M+H]⁺ 454.2, found 453.9.

To a solution of compound 14 (0.004 g, 0.009 mmol) in water (0.030 mL)was added 0.1 mL of 50 mM KH₂PO₄, followed by 4-fluorobenzaldehyde(0.0011 g, 0.009 mmol) in 0.05 mL acetonitrile. The reaction mixture washeated at 90° C. for 10 min, then purified by HPLC (Econosphere C18 10μ,250×10 mm, H₂O/CH₃CN/TFA (65/35/0.1) and compound 12 (0.003 g, 76%) wasobtained. ¹H NMR (400 MHz, D₂O:CD₃CN=3:2) δ7.82 (s, 1H), 7.64 (m, 4H),7.11 (m, 4H), 4.14 (m, 2H), 3.24 (m, 2H), 2.01 (m, 2H), 1.94 (m, 1H),1.80 (m, 2H), 1.52-1.63 (m, 3H), 1.35 (m, 2H). ESI-Mass calcd forC₂₆H₃₁FN₅O₈ [M+H]⁺ 560.2, found 560.1.

Example 14 Synthesis of2-[3-(1-carboxy-5-{4-[N′-(4-[¹⁸F]fluoro-benzylidene)-hydrazino]-benzoylamino}-pentyl)-ureido]-pentanedioicacid (¹⁸F]12)

4-[¹⁸F]fluorobenzaldehyde was synthesized by using a known procedure(Nuclear Medicine and Biology 33 (2006) 677-683). To the crude DMSOsolution of 4-[¹⁸F]fluorobenzaldehyde was added 1-2 mg of compound 14,0.2 mL of 50 mM KH₂PO₄. The vial was closed and heated at 90° C. for 15min. The reaction mixture was then purified by HPLC (Ecomosphere C1810μ, 250×10 mm, H₂O/CH₃CN/TFA [70/30/0.1], 4 mL/min). The radiochemicalyield (decay corrected and based on starting [¹⁸F] fluoride, n=2) was30-55%. The compound 12 decomposed soon after been made. The specificactivity of final compound was not determined.

Example 15 Synthesis of2-{3-[1-carboxy-5-({6-[N′-(4-fluoro-benzylidene)-hydrazino]-pyridine-3-carbonyl}-amino)-pentyl]-ureido}-pentanedioicacid (15)

To a solution of compound 1 (0.040 g, 0.047 mmol) in CH₂Cl₂ (2 mL) wasadded triethylamine (0.020 mL, 0.14 mmol), followed bysuccinimidyl-6-(N′-tert-butoxycarbonyl-hydrazino)-nicotinate (0.020 g,0.057 mmol, Bioconjugate Chem. 1991, 2, 233-336). After stirred, for 1hour at room temperature, the solvent was evaporated. The crude materialwas purified by a silica column using methanol/methylene chloride(10:90) to afford 0.032 g (74%) of compound 16. ¹H NMR (400 MHz, CDCl₃)δ8.54 (s, 1H), 7.90 (m, 1H), 7.17-7.23 (m, 6H), 6.90-7.05 (m, 3H),6.79-6.84 (m, 6H), 6.55 (d, J=8.8 Hz, 2H), 5.79 (m, 2H), 4.94-5.07 (m,6H), 4.38-4.45 (m, 2H), 3.74 (m, 9H), 3.26 (m, 2H), 2.33 (m, 2H), 2.07(m, 1H), 1.85 (m, 1H), 1.68 (m, 1H), 1.18-1.55 (m, 13H). ESI-Mass calcdfor C₄₇H₅₉N₆O₁₃ [M+H]⁺ 915.4, found 914.9.

A solution of TFA/CH₂Cl₂ 1:1 (2 mL) was added to compound 16 (0.032 g,0.035 mmol). The mixture was stirred at room temperature for 1 hour,then concentrated on a rotary evaporator. The crude material waspurified by HPLC (Econosphere C18 10μ, 250×10 mm, H₂O/CH₃CN/TFA(92/8/0.1), 4 mL/min) to afford 0.009 g (45%) of compound 17. ¹H HMR(400 MHz, D₂O) δ8.07-8.40 (m, 2H), 7.00-7.13 (m, 1H), 4.18-4.24 (m, 2H),3.38 (m, 2H), 2.48 (m, 2H), 2.14 (m, 1H), 1.86-1.98 (m, 2H), 1.62-1.65(m, 3H), 1.44 (m, 2H). ESI-Mass calcd for C₁₈H₂₇N₆O₈ [M+H]⁺ 455.2, found455.0.

To a solution of compound 17 (0.005 g, 0.0011 mmol) in water (0.030 mL)was added 50 mM KH₂PO₄ 0.1 mL, followed by 4-fluorobenzaldehyde (0.002g, 0.0016 mmol) in 0.05 mL acetonitrile. The reaction mixture was heatedat 90° C. for 10 min, then purified by HPLC (Econsphere C18 10μ, 250×10mm, H₂O/CH₃CN/TFA (80/20/0.1) and compound 15 (0.002 g, 41%) wasobtained. ¹H NMR (400 MHz, D₂O) δ8.38 (m, 1H), 8.22 (m, 2H), 7.83 (m,2H), 7.20 (m, 3H), 4.26 (m, 2H), 3.41 (m, 2H), 2.52 (m, 2H), 2.11 (m,1H), 1.92 (m, 2H), 1.73 (m, 3H), 1.47 (m, 2H). ESI-Mass calcd forC₂₅H₃₀FN₆O₈ [M+H]⁺ 561.2, found 560.9.

Example 16 Synthesis of2-{3-[1-carboxy-5-({6-[N′-(4-[¹⁸F]fluoro-benzylidene)-hydrazino]-pyridine-3-carbonyl}-amino)-pentyl]-ureido}-pentanedioicacid ([¹⁸F]15)

4-[¹⁸F]fluorobenzaldehyde was synthesized by using a known procedure(Nuclear Medicine and Biology 33 (2006) 677-683). To the crude DMSOsolution of 4-[¹⁸F]fluorobenzaldehyde was added 1-2 mg of compound 16,0.2 ml of 50 mM KH₂PO₄. The vial was closed and heated at 90° C. for 15min. The reaction mixture was then purified by HPLC (Ecomosphere C1810μ, 250×10 mm, H₂O/CH₃CN/TFA [80/20/0.1], 4 mL/min). The radiochemicalyield (decay corrected and based on starting [¹⁸F]fluoride, n=1) was49%.

Example 17

In Vitron Binding.

NAALADase Assay.

NAAG hydrolysis was performed essentially as described previously (26)(27) Briefly, LNCaP cell extracts were prepared by sonication inNAALADase buffer (50 mM Tris [pH 7.4] and 0.5% Triton X-100). Celllysates were incubated with or without inhibitor at 37° C. for 10 min.Following the incubation, the radiolabeled substrateN-acetyl-L-aspartyl-L-(3,4-³H)glutamate (NEN Life Science Products,Boston, Mass.) was added to a final concentration of 30 nM at 37° C. for10-15 min. The reaction was stopped by the addition of an equal volumeof ice-cold 100 mM sodium phosphate and 2 mM EDTA. Products werepartitioned by AG 1-X8 formate resin (Bio-Rad Laboratories, Hercules,Calif.) anion exchange chromatography, eluted with 1 M sodium formate,and quantified by liquid scintillation counting. Inhibition curves weredetermined using semi-log plots and IC₅₀ values determined at theconcentration at which enzyme activity was inhibited by 50%. Assays wereperformed in triplicate with the entire inhibition study being repeatedat least once to confirm affinity and mode of inhibition. Data werecollected during the linear phase of hydrolysis (i.e., <20% cleavage oftotal substrate). Enzyme inhibitory constants (K_(i) values) weregenerated using the Cheng-Prusoff conversion (28). Data analysis wasperformed using GraphPad Prism version 4.00 for Windows (GraphPadSoftware, San Diego, Calif.).

PSMA activity was also determined using a fluorescence-based assayaccording to a previously reported procedure (20). Briefly, lysates ofLNCaP cell extracts were incubated with inhibitor in the presence of 4μM NAAG. The amount of reduced glutamate was measured by incubating witha working solution of the Amplex Red glutamic acid kit (Molecular ProbesInc., Eugene, Oreg., USA). The fluorescence was determined by readingwith a VICTOR3V multilabel plate reader (Parkin Elmer Inc., Waltham,Mass., USA) with excitation at 490 nm and emission at 642 nm.

The NAALADase inhibition assay was undertaken to determine the K_(i)value for 3, 6 and 8 (26). The concentration of each compound was variedfrom 0.01 nM to 1000 nM against a fixed amount of [³H]NAAG (30 nM). TheNAALADase (PSMA) was prepared from LNCaP cell lysates. The percentage ofthe enzymatic cleavage product, [³H]glutamate, produced was measured byscintillation counting and was plotted against the logarithmicconcentration of the compound under study. Linear regression of theresulting data were solved for 50% [³H]glutamate production (50%inhibition) and resulted in K_(i) values of 0.010 nM for 3, 0.256 nM for6 and 0.351 nM for 8 (Table 1). That result is in keeping with othercompounds of this class (30). When these compounds were evaluatedthrough a fluorescence-based inhibition assay as a second check onaffinity, K_(i) values of 3, 6, and 8 were 0.010, 0.194, and 0.557 nM,respectively.

TABLE 1 PSMA in vitro Inhibitory Activities and Calculated ClogD valuescompd K_(i) [nM]^(a) SD^(b) K_(i) [nM]^(c) SD^(b) ClogD 3 0.010 0.0030.010 0.004 −5.16 6 0.256 0.038 0.194 0.134 −5.64 8 0.351 0.257 0.5570.265 −5.88 ^(a)obtained from the NAALADase (radiometric) assay. ^(b)95%confidence interval. ^(c)obtained from a fluorescence-based assay.

Example 18 Modeling of Inhibitors in the Active Site of PSMA

Protein Preparation.

The 3-D coordinates of GCPII for docking studies were prepared as GCPIIcrystal structures in complex with 2-PMPA (PDB ID: 2PVW) or compound 3(PDB ID:3D7H) through a clean-up process implemented in Discovery Studio2.0 (DS 2.0), which can correct for structural disorder, fix bond orderand connectivity of amino acid residues. The CHARMm forcefield that wasapplied to the protein and the binding site for docking studies wasobtained through an automated method by using the option of findingsites from receptor cavities. Two zinc ions and one chloride ion in theactive site were typed as Zn²⁺ and Cl⁻, with formal charges of +2 and−1, respectively.

Docking Studies with CDOCKER.

Docking studies of compounds 3, 6 and 8 were performed with twoconformers of 2PVW using the CDOCKER module implemented in DS 2.0 bymodifying the default settings (Top bits: 20, random conformations: 20,random conformation dynamics steps: 1000, random conformations dynamicstarget temperature: 1000, orientation to refine: 20, maximum badorientations: 800, orientation of vdW energy threshold: 300, simulationheating steps: 2000, heating target temperature: 700, cooling steps:5000, cooling target temperature: 300, Grid extension: 8, ligand partialcharge: CHARMm). The best pose of each ligand with high CDOCKER energywas used for generating the overlay structures (FIGS. 1 and 2) withcrystal ligand 3 (shown in lighter color) from GCPII complex (PDB ID:3D7H). Seven water molecules within 3 Å from crystal ligand 3 wereincluded for docking studies with 3D7H.

The PSMA crystal structure complexed with 3, which was deposited in theprotein data bank (PDB ID: 3D7H) was resolved. Details of PSMAco-crystallized with 3 as well as with other urea-based PSMA inhibitorssuch as DCIT, DCMC and DCFBC, are described by Barinka et al. (39). Aspredicted from modeling studies, only the binding conformation of thearginine patch region was found in the PSMA complex with 3. To elucidatepotential binding modes of the other two compounds (6 and 8), dockingstudies using the 3-D coordinates of 3D7H in the presence or in theabsence of water molecules in the active site were carried out. The bestposes of 3, 6 and 8 from the docking studies using the CDOCKER moduleare shown in FIG. 1, overlaid with crystal ligand, i.e., the compound asit is co-crystallized with PSMA, of 3. As shown in FIG. 1, all of theradionuclide-bearing moieties (4-iodophenyl, 4-fluorophenyl, and5-iodo-3-pyridyl) were located within the arginine patch of the S1binding site. 4-Iodophenyl and 4-fluorophenyl groups protruded deeplywithin the subpocket compared to the 5-iodo-3-pyridyl moiety. CDOCKERscores of the three poses are ordered 3 (80.63)>6 (72.39)>8 (69.78).Aromatic rings of 3, 6 and 8 provide π-π interactions with the guanidinefunctions of Arg 463 and Arg 534, leading to stabilization of the ligandwithin the subpocket. In the case of 8, the nitrogen of the pyridinering enables electrostatic interaction with the carboxylate of Asp 465and hydrogen bonding with one water molecule. Docking results of PSMAwithout water molecules in the active site showed that theradionuclide-bearing moieties of 6 and 8 were outside of the subpocketand protected into the tunnel region (FIG. 2) while the 4-iodophenylgroup of 3 was projected into the subpocket. Based on in vitro PSMAinhibitory activities and molecular modeling studies, it appears thatligand interaction with the subpocket of the S1 binding site contributesmore to binding affinity than interaction with the tunnel region.

Not surprisingly, 3, 6 and 8 assume similar conformations within thePSMA active site. The radionuclide-bearing moiety resides within thearginine patch region of the S1′ binding site in each case, however,that of 8 is not as deep within the pocket (FIG. 1). Compound 3 has beenco-crystallized with PSMA, and the binding conformation for thatcompound shows productive π-π stacking with Arg 463 and Arg 534 of theenzyme, whereas the pyridine moiety of 8 is unable to form a similarlyproductive π-π interaction. However, unlike 3 or 6, 8 is able tointeract with Asp 465 (via the pyridine nitrogen), with Asp 453 (via thecarbonyl oxygen) and a water molecule because the carbonyl group of theradionuclide-bearing moiety points toward the S1 subpocket. Thoseadditional interactions likely offset the less productive π-π bondinggeometry of 8, providing a high-affinity interaction and an imagingagent that gives clear delineation of tumor (FIG. 5). While 6 adopts avery similar conformation within the active site as 3, it binds withsignificantly lower affinity, likely due to additional interactions ofthe iodine within the positively-charged arginine patch for 3. In all,the affinities of 3, 6 and 8 (Table 1) track with predications based onmolecular modeling.

Biodistribution and Imaging

Cell Lines and Mouse Models: PC-3 PIP (PSMA⁺) and PC-3 flu (PSMA⁻) celllines were obtained from Dr. Warren Heston (Cleveland Clinic) and weremaintained as previously described (19). All cells were grown to 80-90%confluence before trypsinization and formulation in Hank's Balanced SaltSolution (BBSS, Sigma, St Louis, Mo.) for implantation into mice.

All animal studies were carried out in full compliance withinstitutional guidelines related to the conduct of animal experiments.Male severe-combined immunodeficient (SCID) mice (Charles RiverLaboratories, Wilmington, Mass.) were implanted subcutaneously with1-5×10⁶ cells forward of each shoulder. PC-3 PIP cells were implantedbehind the left shoulder and PC-3 flu cells were implanted behind theright shoulder. Mice were imaged or used in biodistribution assays whenthe tumor xenografts reached 3-5 mm in diameter.

Ex Vivo Biodistribution and Imaging

Example 19

Compound [¹²⁵I]3.

Xenograft-bearing SCID mice were injected via the tail vein with 74 Bq(2 μCi) of [¹²⁵I]3. Four mice each were sacrificed by cervicaldislocation at 30, 60, 120, 300 min, 12, 24 and 48 hours, p.i. Theheart, lungs, liver, stomach, pancreas, spleen, fat, kidney, muscle,small and large intestines, urinary bladder and PIP and flu tumors werequickly removed. A 0.1 mL sample of blood was also collected. The organswere weighed and the tissue radioactivity measured with an automatedgamma counter (1282 Compugamma CS, Pharmacia/LKB Nuclear, Inc,Gaithersburg, Md.). The percent-injected dose per gram of tissue (%ID/g) was calculated by comparison with samples of a standard dilutionof the initial dose. All measurements were corrected for decay.

Table 2 outlines the ex vivo rodent tissue distribution results of[¹²⁵I]3. The blood, kidney, urinary bladder, spleen and PSMA⁺ PC-3 PIPtumor display high uptake at the initial, 30 min postinjection (p.i.)time point. By 60 min p.i., the urinary bladder displays the highestuptake while the uptake in PSMA⁺ PC-3 PIP tumor also achieves itshighest absolute value. The kidney achieves its maximal uptake at 24 hp.i. The values noted in the kidney are largely due to specific bindingrather than renal clearance, due to the expression of high amounts ofPSMA in the proximal renal tubule (31) (32). Urinary bladder uptakerepresents excretion at all time points, i.e., there was no specificbinding to bladder wall. Tumor uptake demonstrates a high degree ofspecificity represented by the PIP:flu ratio of 10:1 at 60 min andrising to 140:1 at 48 h. Radiopharmaceutical uptake within tumorrelative to other organs also increases with time. Site-specificblockade studies were not performed as they were perceived to beredundant in light of the use of engineered (PC-3 PIP and PC-3 flu)tumors to determine binding specificity.

TABLE 2 Ex vivo biodistribution of [¹²⁵I]3 in tumor-bearing mice.^(a,b)Organ 30 min 60 min 2 h 5 h 12 h 24 h 48 h blood 0.9 ± 0.8 0.6 ± 0.2 0.3± 0.1 0.4 ± 0.2  0.1 ± 0.03 ND ND (10)  (22)  (36)  (31)  (125)  heart2.7 ± 0.9 1.9 ± 0.3 1.4 ± 0.7 0.9 ± 0.2 0.5 ± 0.2 0.5 ± 0.2 0.1 ± 0.1(3) (7) (8) (14)  (25)  (28)  (100)  lung 5.8 ± 2.4 4.5 ± 0.5 3.6 ± 2.83.5 ± 0.6 2.6 ± 0.8 1.1 ± 0.8  0.5 ± 0.04   (1.5) (3) (3)   (3.5) (5)(12)  (22)  liver 7.7 ± 3.1 7.5 ± 0.8 5.9 ± 3.5 4.5 ± 1.0 1.4 ± 0.2 1.4± 0.6 0.7 ± 0.1 (1) (2) (2) (3) (9) (9) (16)  stomach 1.5 ± 0.9 1.5 ±0.3 1.6 ± 1.5  0.8 ± 0.03  0.3 ± 0.06 0.7 ± 0.6 0.4 ± 0.2 (6) (9) (8)(15)  (39)  (18)  (25)  pancreas 2.0 ± 0.3 2.3 ± 0.3 2.0 ± 0.7 1.6 ± 0.50.6 ± 0.2 1.2 ± 0.7 1.1 ± 0.6 (4) (6) (6) (8) (21)  (11)  (10)  spleen83 ± 8  141 ± 14  104 ± 43  119 ± 9  69 ± 39 39 ± 6   22 ± 8.6   (0.1)  (0.1)   (0.1)   (0.1)   (0.2)   (0.3)   (0.5) fat 4.5 ± 1.1 5.6 ± 1.06.2 ± 0.8 6.8 ± 1.3 3.8 ± 0.8 1.6 ± 1.9 2.8 ± 0.7 (2) (2) (2) (2) (3)(8) (4) kidney 119 ± 15  121 ± 17  111 ± 34  132 ± 12  169 ± 29  234 ±140 101 ± 30    (0.1)  (0.1)   (0.1)   (0.1)   (0.1)   (0.1)   (0.1)muscle 2.7 ± 2.4 0.8 ± 0.2 0.6 ± 0.2 0.4 ± 0.1 1.0 ± 0.1 0.4 ± 0.1 0.25± 0.03 (3) (17)  (21)  (31)   (12.5) (33)  (44)  small int. 4.9 ± 1.93.8 ± 0.4 1.5 ± 0.3 1.5 ± 0.2 1.0 ± 0.4 0.25 ± 0.1  0.1 ± 0.1 (2)  (3.5) (8) (8)  (12.5) (54)  (110)  large int. 1.4 ± 0.6 1.0 ± 0.2 0.6± 0.1 0.7 ± 0.6 0.6 ± 0.2 1.6 ± 1.9 0.15 ± 0.1  (6)  (13.5) (21)  (17) (21)  (9) (73)  bladder 5.2 ± 1.7 6.1 ± 0.8 4.0 ± 2.6 3.0 ± 1.7 0.8 ±0.3 0.2 ± 0.2  0.3 ± 0.04 (2) (2) (3) (4) (16)  (64)  (37)  PC-3 PIP 8.8± 4.7 13.5 ± 2.1  11.8 ± 5.6  12.4 ± 6.4  12.5 ± 4.8  13.4 ± 5.1  11.0 ±0.2  PC-3 flu 1.8 ± 1.0 1.2 ± 0.3 0.7 ± 0.3  0.6 ± 0.05 0.3 ± 0.1  0.1 ±0.06 0.08 ± 0.06 PIP:flu 5 11  18  19  48  131  140  ^(a)Values are in %ID/g ± SD; ND = not determined; n = 4 except at 48 h where n = 3. Int. =intestine. ^(b)Pip tumor:organ ratios are in parentheses

Example 20

Compound [¹⁸F]6.

Ex vivo biodistribution proceeded as for [¹²³I]3 with the followingexceptions: Mice were injected with 3.7 MBq (100 [μCi) of [¹⁸F]6 anduptake times were 30, 60, 120 and 300 minutes p.i.

Table 3 illustrates tissue uptake of [¹⁸F]6. This radiopharmaceuticalalso displayed rapid, specific uptake within PSMA⁺ PIP tumors(8.58±3.09% ID/g at 30 min. p.i.) and kidney (72.05±3.19% ID/g at 30min. p.i.). Uptake in and washout from nonspecific tissues was low andfast, respectively. Only the liver and spleen display uptake values thatrival those seen in PIP tumor. The spleen exhibits the highest uptake ofany nonspecific tissue (12.67±0.36% ID/g at 30 min. p.i.), perhaps dueto rise presence of GCPIII, a close homolog of GCPII/PSMA (33). It isspeculated that [¹⁸F]6 may bind to GCPIII as well as to GCPII, asseveral other PSMA ligands have been shown to do so (34).

TABLE 3 Ex vivo biodistribution of [¹⁸F]6 in tumor-bearing mice.^(a,b)Organ 30 min 60 min 2 h 5 h blood 2.5 ± 1.7 0.7 ± 0.5 0.4 ± 0.2 0.03 ±0.00 (3)  (9)  (9) (117)  heart 0.8 ± 0.1  0.2 ± 0.02 0.15 ± 0.05 0.03 ±0.01 (11)  (35) (25) (117)  lung 1.7 ± 0.3 0.5 ± 0.1 1.0 ± 0.9 0.1 ± 0.1(5) (13)  (4) (35) liver 8.7 ± 1.8 5.8 ± 0.6 11.7 ± 7.0  1.0 ± 0.6 (1) (1)   (0.3)   (3.5) stomach 0.8 ± 0.1 0.25 ± 0.1  0.3 ± 0.1 0.04 ± 0.02(11)  (26) (13) (88) pancreas 0.8±0.1 0.3 ± 0.1 0.15 ± 0.03 0.05 ± 0.03(11)  (21) (25) (70) spleen 12.7 ± 0.4  7.2 ± 1.6 4.4 ± 1.2 1.0 ± 0.6 (0.7)  (1)  (1)   (3.5) kidney 72 ± 3  48 ± 5  29 ± 12 14 ± 9   (0.1)  (0.1)   (0.1)   (0.3) muscle 2.4 ± 3.1 0.5 ± 0.7 0.2 ± 0.1 0.1 ± 0.1(4) (13) (19) (35) small int. 1.7 ± 0.7 0.6 ± 0.2 0.3 ± 0.2  0.1 ± 0.04(5) (11) (12) (35) large int. 1.2 ± 0.8 0.5 ± 0.1 0.2 ± 0.1 0.6 ± 0.6(7) (13) (19)  (6) bladder 6.8 ± 3.5 17 ± 21 11 ± 8  5.2 ± 8.9 (1)  (0.4)   (0.3)   (0.7) PC-3 PIP 8.6 ± 3.1 6.4 ± 0.9 3.7 ± 1.2 3.5 ± 2.3PC-3 flu 0.8 ± 0.3  0.3 ± 0.05 0.25 ± 0.1  0.1 ± 0.1 PIP:flu 11  21 1535 ^(a)Values are in % ID/g ± SD, n = 4, int. = intestine. ^(b)Piptumor:organ ratios are in parentheses.

Example 21

Compound [¹²⁵I]8.

PC-3 PIP and PC-3 flu xenograft-bearing SCID mice were injected via thetail vein with 74 KBq (2 μCi) of [¹²⁵I]8. Four mice each were sacrificedby cervical dislocation at 30, 60, 240 min., 8 and 24 hours p.i. Theheart, lungs, liver, stomach, pancreas, spleen, fat, kidney, muscle,small and large intestines, urinary bladder and PIP and flu tumors werequickly removed. A 0.1 mL sample of blood was also collected. All theorgans were weighed and the tissue radioactivity measured with anautomated gamma counter (1282 Compugamma CS, Pharmacia/LKB Nuclear, Inc,Gaithersburg, Md.). The % ID/g was calculated by comparison with samplesof a standard dilution of the initial dose. All measurements werecorrected for decay.

Table 4 outlines the ex vivo rodent tissue distribution results of[¹²⁵I]8. The liver, spleen, kidney and PSMA⁺ PC-3 PIP tumors displayedhigh uptake at the initial, 30 min p.i. time point. By 60 min p.i., thekidney displayed the highest uptake while that in PSMA⁺ PC-3 PIP rumorremained steady, showing a similar value to that at 30 min. By 24 h,there was complete clearance of radioactivity from the individual,nontarget organs. The values noted in the kidney are largely due tospecific binding rather than renal clearance, as for the otherradiopharmaceuticals discussed above. Urinary bladder uptake representedexcretion at all time points, i.e., there was no specific binding tobladder wall, while tumor uptake demonstrated a high degree ofspecificity represented by the PIP:flu uptake ratio of 18:1 at 30 min,rising to 48:1 at 24 h. As for [¹²⁵I]3, the radiopharmaceutical uptakewithin tumor relative to other organs increases with time.

TABLE 4 Ex vivo biodistribution of [¹²⁵I]8 in tumor-bearing mice.^(a,b)Organ 30 min 60 min 4 h 8 h 24 h blood 2.5 ± 1.4 1.1 ± 0.5 0.25 ± 0.2 0.06 ± 0.01 0.00 ± 0.00 (6) (11) (17) (23) heart 0.9 ± 0.2 0.4 ± 0.30.06 ± 0.02 0.02 ± 0.01 0.00 ± 0.00 (16)  (30) (20) (70) lung 2.7 ± 3.22.3 ± 1.0 0.3 ± 0.1  0.1 ± 0.05 0.01 ± 0.00 (5)  (5) (14) (12) (9) liver8.2 ± 1.2 9.7 ± 1.2 4.8 ± 0.9 1.6 ± 0.4 0.04 ± 0.03 (2)  (1)  (1)  (1)(2) stomach 0.8 ± 0.3 0.5 ± 0.3  0.1 ± 0.05 0.06 ± 0.02 0.01 ± 0.01(18)  (23) (42) (23) (9) pancreas 0.9 ± 0.2 0.8 ± 0.3 0.3 ± 0.3 0.03 ±0.00 0.00 ± 0.00 (15)  (16) (14) (47) spleen 26 ± 12 13.0 ± 6.8  1.25 ±0.4  0.5 ± 0.2 0.03 ± 0.02  (0.5)  (1)  (3)  (3) (3) fat 3.5 ± 0.2 1.4 ±0.5 0.04 ± 0.1  0.2 ± 0.2 0.00 ± 0.01 (4)  (8) (105)   (7) kidney 160 ±27  205 ± 46  71 ± 27 24 ± 10 0.97 ± 0.94  (0.1)   (0.06)   (0.06)  (0.06)  (0.1) muscle 1.9 ± 2.4 0.9 ± 0.7 0.2 ± 0.3 0.1 ± 0.1 0.00 ±0.00 (7) (13) (21) (14) small int. 0.8 ± 0.2 1.1 ± 1.3  0.2 ± 0.05  0.1± 0.02 0.01 ± 0.01 (18)  (11) (21) (14) (9) Large int. 0.9 ± 0.4 0.6 ±0.3 0.2 ± 0.1 0.1 ± 0.1 0.00 ± 0.00 (16)  (21) (21) (14) bladder 1.9 ±0.4 4.8 ± 4.9 7.0 ± 3.6 2.8 ± 2.1 0.05 ± 0.01 (8)   (2.5)   (0.6)  (0.5) (2) PC-3 PIP 14.2 ± 9.5  12.1 ± 4.9  4.2 ± 1.5 1.4 ± 0.4 0.09 ±0.04 PC-3 flu 0.8 ± 0.1  0.6 ± 0.25  0.1 ± 0.02 0.03 ± 0.01 0.00 ± 0.00PIP:flu 18  20 42 47 ^(a)Values are in % ID/g ± SD; n = 4; int. =intestine. ^(b)Pip tumor:organ ratios are in parentheses.

Regarding ex vivo biodistribution, 3 demonstrates only about twice theturner uptake at 1 h p.i. as 6, while its affinity is about twenty-fivetimes higher. The target to nontarget (PIP:flu) ratio for 6, however, ishigher than for 3, reflecting lower nonspecific binding. Those target tonontarget ratios rise to approximately 140 at 48 hours p.i. for 3 and to31 at 5 hours p.i. for 6. Compound 8 differs from 3 in that the aromaticring is a pyridine and the iodine is substituted at the three-position.At one hour p.i. 8 demonstrated a similarly high tumor uptake(12.05±4.92% ID/g) compared to 3, but had a much higher target tonontarget (PIP:flu) ratio at that lime (22), which rose to 48.3 at 24 hp.i. Interestingly, the affinity of 8 is the lowest among the threecompounds tested (Table 1), however, it provides the highest target tonontarget ratio at 1 h pi. As demonstrated in previous work with^(99m)Tc-labeled compounds of this series, it is again shown that thereis not a clear relationship between affinity and in vivo tumor uptakeselectivity. Notably, all of these affinities are quite high and thetumors are clearly delineated (FIGS. 3-5).

In Vivo Biodistribution and Imaging

Example 22

Compound [¹²⁵I]3.

A single SCID mouse implanted with both a PC-3 PIP and a PC-3 fluxenograft was injected intravenously with 37 MBq (1 mCi) of [¹²⁵I]3 insaline. At 4 and 6 h p.i. the mouse was anesthetized with isoflurane andmaintained under 1% isoflurane in oxygen. The mouse was positioned onthe X-SPECT (Gamma Medica, Northridge, Calif.) gantry and was scannedusing two low energy, high-resolution pinhole collimators (Gamma Medica)rotating through 360° in 6° increments for 45 seconds per increment. Allgamma images were reconstructed using Lunagem software (Gamma Medica,Northridge, Calif.). Immediately following SPECT acquisition, the micewere then scanned by CT (X-SPECT) over a 4.6 cm field-of-view using a600 μA, 50 kV beam. The SPECT and CT data were then coregistered usingthe supplier's software (Gamma Medica, Northridge, Calif.) and displayedusing AMIDE (http://amide.sourceforge.net/). Data were reconstructedusing the Ordered Subsets-Expectation Maximization (OS-EM) algorithm.

FIG. 3 shows a SPECT-CT image of radiopharmaceutical uptake at 4 h p.i.Note the intense uptake in the PC-3 PIP and absence of uptake in thePC-3 flu tumor. Thyroid uptake of the radiotracer indicates the presenceof free [¹²⁵I]iodide due to deiodination by dehalogenases (35) (36). Theamount of free [¹²⁵I]iodide, however, is small in comparison to theamount of PC-3 PIP tumor uptake (thyroid:muscle=2; PIPtumor:thyroid=12.5). The small amount of radiopharmaceutical uptake seenin the liver, with no concurrent gastrointestinal uptake, is likely dueto the hydrophilic nature of [¹²⁵I]3 (ClogD=−5.16 at pH 7.4).

Example 23

Compound [¹⁸F]6.

In vivo PET-CT: A SCID mouse bearing subcutaneous PC-3 PIP and PC-3 fluxenografts was anesthetized using 3% isoflurane in oxygen for inductionand 1.5% isoflurane in oxygen at 0.8 L/min flow for maintenance andpositioned prone on the gantry of a GE explore Vista small animal PETscanner (GE Healthcare, Milwaukee, Wis.). The mouse was injectedintravenously with 7.4 MBq (200 μCi) of [¹⁸F]6 followed by imageacquisition using the following protocol: The images were acquired as apseudodynamic scan, i.e., a sequence of successive whole-body imagesacquired in three bed positions for a total of 90 min. The dwell time ateach position was 5 minutes, such that a given bed position (or mouseorgan) was revisited every 15 min. An energy window of 250-700 keV wasused. Images were reconstructed using the FORE/2D-OSEM method (2iterations, 16 subsets) and included corrections for radioactive decay,scanner dead time and scattered radiation.

FIG. 4 shows the averaged results of the dynamic scan from 94-120 minp.i. The uptake pattern for [¹⁸F]6 is very similar to that seen for[¹²⁵I]3: easily observed within PIP tumor, none within flu tumor, highrenal uptake and a modest degree of liver uptake. That result wasexpected due to similar ClogD values for [¹⁸F]6 (−5.64 at pH 7.4) and[¹²⁵I]3. As for [¹²⁵I]3, the urinary bladder is visualized due to thecontinually accumulating presence of radioactive urine, however,specific binding to bladder wall was not demonstrated.

Example 24

Compound [¹²⁵I]8.

A single SCID mouse implanted with a LNCaP xenograft was injectedintravenously with 37 MBq (1 mCi) of [¹²⁵I]8 in saline. At 4 h p.i., themouse was anesthetized with isoflurane and maintained under 1%isoflurane in oxygen. The mouse was positioned on the X-SPECT (GammaMedica, Northridge, Calif.) gantry and was scanned using two low energy,high-resolution pinhole collimators (Gamma Medica) rotating through 360°in 6° increments for 45 seconds per increment. All gamma images werereconstructed using Lunagem software (Gamma Medica, Northridge, Calif.).Immediately following SPECT acquisition, the mice were then scanned byCT (X-SPECT) over a 4.6 cm field-of-view using a 600 μA, 50 kV beam. TheSPECT and CT data were then coregistered using the supplier's software(Gamma Medica, Northridge, Calif.) and displayed using AMIDE(http://amide.sourceforge.net/). Data were reconstructed using theOrdered Subsets-Expectation Maximization (OS-EM) algorithm.

FIG. 5 shows a SPECT-CT Image of radiopharmaceutical uptake at 4 h pi.Tumor uptake and retention was high, with slow washout, while thewashout of [¹²⁵I]8 from nontarget tissue was rapid.

Comparative Data for Target/Nontarget Ratios for Phenyl Versus PyridineAnalogs.

Target/non-target ratios are given for compound 3 (4-iodobenzoylderivative) at 5 hours post injection and compound 8(3-iodo-5-carboxyl-pyridyl derivative) at 4 hour postinjection. Thetarget/non-target ratios are shown in Table 5 below.

TABLE 5 Cmpd. 3 Cmpd. 8 Tumor (T)/organ (5 hours post injection) (4hours post injection) T/blood 31 17 T/heart 14 20 T/Lung 3.5 14 T/liver3 1 T/stomach 15 42 T/Pancreas 8 14 T/Spleen 0.1 3 T/fat 2 105 T/kidney0.1 0.1 T/muscle 31 21 T/small Intest 8 21 T/large Intest 17 21

It appears that the improved target/non-target ratios for 8 versus 3 isdue to the faster non-target clearance of 8 even though the bloodclearance of each are comparable and 3 has higher tumor retentionespecially at later time points. Compound 3 is more lipophilic than 8and has higher uptake in fat. The retention of 3 in fat could beproviding a slow release of 3 for uptake in tumor and normal organs.

The high and prolonged tumor and kidney (PSMA rich in mice) uptake of 3is due to this compounds tight binding to PSMA. The 4-iodophenyl moietyresides in a hydrophobic pocket accessory to the S1 binding site andprovides additional hydrophobic-hydrophobic interactions (39). Pyridineanalogs are more polar than 3 so they should have reducedhydrophobic-hydrophobic interactions in this binding site.

Compound 6 has even better target/nontarget ratios than compound 8.Therefore the background clearance of the more polar pyridine analog of6 should give even better tumor-non-target ratios.

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The invention claimed is:
 1. A composition comprising a compound of the formula:

or a pharmaceutically acceptable salt thereof.
 2. The composition of claim 1, wherein the pharmaceutically acceptable salt is a sodium salt, potassium salt, calcium salt, magnesium salt, or quaternary ammonium salt.
 3. A kit comprising a packaged pharmaceutical composition comprising a pharmaceutically acceptable carrier and the composition of claim
 1. 4. A method of imaging one or more cells, organs, or tissues comprising exposing the one or more cells, organs, or tissues to, or administering to a subject, a detectably effective amount of the composition of claim 1 and imaging said one or more cells, organs, or tissues.
 5. The method of claim 4, wherein the one or more tissues comprise a prostate tissue.
 6. The method of claim 4, wherein the one or more tissues comprise a kidney tissue, brain tissue, vascular tissue, tumor tissue, endometrium, schwannoma tissue, or Barrett's esophagus tissue.
 7. The method of claim 4, wherein the one or more cells comprise cancer cells selected from lung cancer cells, renal cancer cells, glioblastoma cells, pancreas cancer cells, bladder cancer cells, sarcoma cells, melanoma cells, breast cancer cells, colon cancer cells, esophageal cancer cells, and stomach cancer cells.
 8. The method of claim 7, wherein the cancer cells express prostate-specific membrane antigen (PSMA) in the neovasculature.
 9. The method of claim 4, wherein the one or more cells comprise prostate cancer cells.
 10. The method of claim 4, wherein the one or more cells comprise germ cells, pheochromocytoma cells, or vascular cells undergoing angiogenesis.
 11. A method of treating a tumor comprising administering to a subject a therapeutically effective amount of the composition of claim
 1. 12. The method of claim 11, wherein the tumor is prostate cancer. 